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EHD1 and RUSC2 Control Basal Epidermal Growth Factor Receptor Cell Surface Expression and Recycling
Open accessAbstract
Epidermal growth factor receptor (EGFR) is a prototype receptor tyrosine kinase and an oncoprotein in many solid tumors. Cell surface display of EGFR is essential for cellular responses to its ligands. While postactivation endocytic trafficking of EGFR has been well elucidated, little is known about mechanisms of basal/preactivation surface display of EGFR. Here, we identify a novel role of the endocytic regulator EHD1 and a potential EHD1 partner, RUSC2, in cell surface display of EGFR. EHD1 and RUSC2 colocalize with EGFR in vesicular/tubular structures and at the Golgi compartment. Inducible EHD1 knockdown reduced the cell surface EGFR expression with accumulation at the Golgi compartment, a phenotype rescued by exogenous EHD1. RUSC2 knockdown phenocopied the EHD1 depletion effects. EHD1 or RUSC2 depletion impaired the EGF-induced cell proliferation, demonstrating that the novel, EHD1- and RUSC2-dependent transport of unstimulated EGFR from the Golgi compartment to the cell surface that we describe is functionally important, with implications for physiologic and oncogenic roles of EGFR and targeted cancer therapies.
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Written from the paper’s full text.
TL;DR
This paper identifies EHD1 and RUSC2 as positive regulators of basal, ligand-unstimulated EGFR transport from the Golgi compartment to the cell surface in mammary epithelial cells and other cell types. Depletion of either protein reduces surface EGFR, impairs EGF-induced signaling and proliferation, and causes EGFR retention in a Golgi-associated compartment.
Key findings
- EHD1 knockdown reduced total and cell surface EGFR under ligand-free conditions, with EGFR accumulating in a GM130+ Golgi compartment; exogenous EHD1-GFP rescued total EGFR levels.
- RUSC2 knockdown phenocopied EHD1 depletion: it impaired total EGFR accumulation after EGF withdrawal, decreased cell surface EGFR by FACS, and reduced EGF-induced proliferation using two independent shRNAs.
- EHD1 and RUSC2 colocalized with EGFR in vesicular/tubular structures and at the Golgi; RUSC2 NPF motifs bound the EHD1 EH domain in a fluorescence polarization assay, but no co-IP was detected between full-length RUSC2 and EHD1 or between EHD1 and EGFR.
- EHD1 knockdown reduced EGF-induced EGFR phosphorylation and proliferation; RUSC2 knockdown also significantly reduced EGF-induced proliferation.
Methods
- Cell systems included 16A5 HPV16 E6/E7-immortalized human mammary epithelial cells, EHD1-floxed and EHD1-null MEFs, MDA-MB-231 breast cancer cells, S2013 pancreatic cancer cells, and HEK-293T cells; Dox-inducible shRNA/siRNA knockdown and EHD1-GFP rescue were used.
- Assays included Western blotting, FACS for surface EGFR and MHC-I, confocal immunofluorescence with GM130/Rab11 markers, [35S]methionine-cysteine pulse-chase, co-immunoprecipitation, GST pulldown, fluorescence polarization competition, and CellTiter-Glo proliferation.
- Statistical comparisons used Student’s t test with means ± SEM and P ≤ 0.05 considered significant.
Limitations
- Stated: the impact of EHD1 depletion was modest in MEFs and other cell systems; the authors suggest this may reflect EGFR dependence of 16A5 cells or redundancy with other EHD family members, noting EHD4 increases in EHD1-depleted MEFs and that double EHD1/EHD4 depletion should be tested.
- Stated: the anti-RUSC2 antibody was suboptimal for imaging endogenous RUSC2, and no physical interaction was detected between EHD1 and RUSC2 or between EHD1 and EGFR, leaving the precise biochemical mechanism of their cooperation unresolved.
- Evident: most mechanistic experiments rely on the 16A5 cell line, with no in vivo tumor or animal model; the MHC-I surface assay did not detect an EHD1 effect under these conditions despite known EHD1 roles in MHC-I recycling, indicating context- or assay-dependent limitations.
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- Findingown data§ ABSTRACT
RUSC2 knockdown phenocopies the EHD1 depletion effects.
“RUSC2 knockdown phenocopied the EHD1 depletion effects.”
Quote in the paperSubstantial claim - Findingown data§ RESULTS
EHD1 knockdown 16A5 cells exhibit substantially reduced EGFR phosphorylation upon EGF stimulation.
“EHD1 KD 16A5 cells exhibited substantially reduced EGFR phosphorylation upon stimulation with EGF, as visualized by phosphotyrosine immunoblotting of cell lysates (Fig. 6A and B).”
Quote in the paperSubstantial claim - Findingown data§ RESULTS
EHD1 knockdown reduces total EGFR levels in 16A5 MECs under ligand-free culture.
“Total EGFR levels measured by Western blotting were reduced in EHD1 KD cells relative to levels in controls, as seen in representative blots and quantified data from three independent experiments (Fig. 1E and F).”
Quote in the paperSubstantial claim - Findingown data§ RESULTS
EHD1 knockdown reduces total EGFR levels in 16A5 MECs under steady-state culture.
“Under conditions of EHD1 KD, we observed a steady reduction in total EGFR levels over the course of the experiment (Fig. 1C and D).”
Quote in the paperSubstantial claim - Findingown data§ RESULTS
EHD1 knockdown reduces cell surface EGFR levels on 16A5 MECs under ligand-free culture.
“Correlating with the depletion of total levels of EGFR, the surface EGFR levels were reduced in EHD1 KD cells relative to levels of the controls (Fig. 2A and B).”
Quote in the paperSubstantial claim
- Findingown data§ ABSTRACT
The reduction in cell surface EGFR expression with Golgi accumulation caused by EHD1 knockdown is rescued by exogenous EHD1.
“Inducible EHD1 knockdown reduced the cell surface EGFR expression with accumulation at the Golgi compartment, a phenotype rescued by exogenous EHD1.”
Quote in the paperSubstantial claim - Findingown data§ RESULTS
RUSC2 has two potential EHD1 EH domain-binding NPF motifs that bind with moderate affinity to the EH domain of EHD1 in vitro.
“We noted two potential EHD1 EH domain-binding NPF motifs in RUSC2 (amino acids 43-NPFCPPELG-51 and 101-NPFLLQEGV-109) which bound with moderate affinity to the EH domain of EHD1 in an in vitro fluorescence polarization-based competition assay using a labeled peptide derived from MICAL-like 1 protein (amino acids 425-NPFEEEEED-433) as a probe (Fig. 7A).”
Quote in the paperNot a substantial claim - Findingown data§ Abstract
EHD1 and RUSC2 colocalize with EGFR in vesicular/tubular structures and at the Golgi compartment.
“EHD1 and RUSC2 colocalize with EGFR in vesicular/tubular structures and at the Golgi compartment.”
- Also stated in: § INTRODUCTION · boosted
“Here, we show that EHD1 and RUSC2 show an overlapping subcellular localization and function as positive regulators of the transport of unstimulated EGFR from the Golgi compartment to the cell surface to help maintain an activation-ready cell surface pool of EGFR on the plasma membrane.”
Quote in the paperSubstantial claim - Also stated in: § INTRODUCTION · boosted
- Findingown data§ RESULTS
EHD1 and EGFR colocalize in intracellular compartments.
“Thus, while EHD1 and EGFR do not appear to physically interact, they colocalize in intracellular compartments, consistent with a role of EHD1 in the intracellular traffic of EGFR.”
Quote in the paperSubstantial claim - Findingown data§ RESULTS
EHD1 knockdown moderately reduces EGFR accumulation after serum and EGF deprivation in MDA-MB-231 cells.
“Moderate EHD1 dependence of the accumulation of EGFR after serum and EGF deprivation was also seen when we compared control shRNA and EHD1 shRNA-expressing versions of a triple-negative breast cancer cell line, MDA-MB-231 (Fig. 3C and D)”
Quote in the paperSubstantial claim - Findingown data§ RESULTS
EHD1 and EGFR colocalize in intracellular vesicular structures in 16A5 cells.
“EHD1+/EGFR+ vesicles were observed as sparse puncta in DMSO-treated cells (Fig. 4A); however, a clear perinuclear EHD1 and EGFR colocalization was observed in monensin-treated cells (Fig. 4B).”
Quote in the paperSubstantial claim - Findingown data§ ABSTRACT
EHD1 or RUSC2 depletion impairs EGF-induced cell proliferation.
“EHD1 or RUSC2 depletion impaired the EGF-induced cell proliferation, demonstrating that the novel, EHD1- and RUSC2-dependent transport of unstimulated EGFR from the Golgi compartment to the cell surface that we describe is functionally important, with implications for physiologic and oncogenic roles of EGFR and targeted cancer therapies.”
Quote in the paperSubstantial claim - Findingown data§ RESULTS
The perinuclear compartment where EGFR accumulates in EHD1 knockdown cells is the Golgi compartment, based on GM130 costaining.
“This perinuclear compartment was identified as the Golgi compartment, based on GM130+ costaining (Fig. 5B).”
Quote in the paperSubstantial claim - Findingown data§ RESULTS
Upon EGF removal, EGFR in control and EHD1-GFP rescue 16A5 cells progressively localizes to the cell surface.
“Upon EGF removal from the medium, EGFR in the control cell line (no Dox treatment) and in the EHD1-GFP rescue cell line (treated with Dox) progressively localized to the cell surface, as expected.”
Quote in the paperSubstantial claim - Findingown data§ RESULTS
EHD1 knockdown results in EGFR retention in the Golgi apparatus.
“EHD1 knockdown results in EGFR retention in the Golgi apparatus.”
Quote in the paperSubstantial claim - Findingown data§ RESULTS
RUSC2 is expressed in 16A5 cells.
“we found that RUSC2 was expressed in 16A5 cells.”
Quote in the paperNot a substantial claim - Findingown data§ RESULTS
MHC-I cell surface levels are comparable in EHD1 knockdown and control 16A5 cells under serum- and EGF-deprived conditions.
“However, MHC-I cell surface levels were comparable in EHD1 KD and control cells.”
Quote in the paperSubstantial claim - Findingown data§ RESULTS
A pool of EHD1 and EGFR colocalizes at the Golgi compartment in 16A5 cells.
“Three-color confocal immunofluorescence staining of endogenous EGFR and the Golgi marker GM130 with ectopically expressed EHD1-DsRed demonstrated that a pool of EHD1 and EGFR was colocalized at the Golgi compartment (Fig. 4E and F).”
Quote in the paperSubstantial claim - Findingown data§ RESULTS
Very little surface-labeled EGFR accumulates in the same GM130/Rab11 compartment in control cells.
“Very little surface-labeled EGFR was seen to accumulate in the same compartment in control cells (Fig. 5D).”
Quote in the paperSubstantial claim - Findingown data§ RESULTS
16A5, MDA-MB-231, and S2013 cells express EHD1 as well as other EHD proteins.
“16A5 as well as other cell lines, including the triple-negative breast cancer cell line MDA-MB-231 and the pancreatic cancer cell line S2013 used in this work, express EHD1 as well as other EHD proteins (Fig. 1B)”
Quote in the paperNot a substantial claim - Findingown data§ RESULTS
EHD1-null MEFs show a diminished capacity for recovery of total EGFR levels over time compared to control MEFs, although the impact is modest.
“we observed a diminished capacity for the recovery of total EGFR levels over time in EHD1-null MEFs compared to levels in control MEFs although the impact was modest (Fig. 3A and B).”
Quote in the paperSubstantial claim - Findingown data§ RESULTS
EGFR prelabeled at the cell surface accumulates in a GM130/Rab11 double-positive perinuclear compartment in EHD1 knockdown cells.
“we observed that EGFR prelabeled at the cell surface accumulated in a GM130/Rab11 double-positive, perinuclear compartment in EHD1 KD cells, as also seen in cells that were not prelabeled on the cell surface (Fig. 5D).”
Quote in the paperSubstantial claim - Findingown data§ RESULTS
RUSC2 knockdown significantly reduces EGF-induced cell proliferation relative to control shRNA cells.
“RUSC2 KD led to a significant reduction in EGF-induced cell proliferation relative to the level in control shRNA cells (Fig. 8E).”
Quote in the paperSubstantial claim - Findingown data§ DISCUSSION
The impact of EHD1 depletion on EGFR levels was also observed in multiple other cell types, including MEFs with conditional EHD1 deletion, although the impact was modest in these other cell systems.
“The impact of EHD1 depletion was also observed in multiple other cell types, including MEFs in which the endogenous floxed EHD1 was conditionally deleted (Fig. 3), albeit the impact was modest in these other cell systems.”
Quote in the paperNot a substantial claim - Findingown data§ DISCUSSION
EHD1 depletion did not significantly impact MHC-I cell surface expression under the ligand-free culture conditions used for EGFR analyses.
“Notably, however, we did not observe a significant impact of EHD1 depletion on MHC-I cell surface expression when this was examined under the same conditions as we used for analyses of EGFR (ligand-free culture), suggesting that our findings may be more relevant to RTKs whose cell surface pools are subject to rapid ligand-induced degradation and require efficient surface delivery through new syn”
Quote in the paperSubstantial claim - Findingown data§ RESULTS
Upon EHD1 knockdown, EGFR, RUSC2, and GM130 show significant three-color colocalization.
“we observed significant three-color colocalization between EGFR, RUSC2, and GM130 upon EHD1 KD (Fig. 7E).”
Quote in the paperSubstantial claim - Findingown data§ DISCUSSION
RUSC2 is not required for EHD1 localization in the Golgi compartment.
“Unlike the recently described EHD1 recruitment to membranes via Rab35 effector MICAL-L1 (79), we did not find any requirement of RUSC2 for EHD1 localization in the Golgi compartment (Fig. 5B), suggesting that they function in a common trafficking pathway but independent of an interaction.”
Quote in the paperSubstantial claim - Findingown data§ RESULTS
RUSC2 and EHD1 colocalize in intracellular vesicles and tubular structures.
“Colocalization was observed when either endogenous EHD1 was immunostained along with exogenous HA-RUSC2 (Fig. 7D, top panel) or when HA-RUSC2 and EHD1-DsRed were examined (Fig. 7D, bottom panel).”
- Also stated in: § DISCUSSION · boosted
“RUSC2 displayed strong colocalization with EHD1 on tubular and vesicular structures, which included the Golgi compartment (Fig. 7D and E).”
Quote in the paperSubstantial claim - Also stated in: § DISCUSSION · boosted
- Findingown data§ DISCUSSION
A portion of the Golgi compartment-localized EGFR is derived from the pool internalized from the cell surface.
“we tested and found support for the idea that a portion of the Golgi compartment-localized EGFR is derived from the pool internalized from the cell surface (Fig. 5A).”
Quote in the paperSubstantial claim - Findingown data§ DISCUSSION
RUSC2 knockdown led to a phenotype that mimicked that of EHD1 knockdown.
“Importantly, RUSC2 KD led to a phenotype that mimicked that of EHD1 KD (Fig. 8).”
Quote in the paperSubstantial claim - Findingnone given§ DISCUSSION
EHD4 levels are substantially increased in MEFs with EHD1 depletion.
“Notably, EHD4 levels are substantially increased in MEFs with EHD1 depletion, and future analyses using MEFs with double EHD1/EHD4 depletion should be instructive in this regard.”
Quote in the paperNot a substantial claim - Interpretationown data§ ABSTRACT
The EHD1- and RUSC2-dependent transport of unstimulated EGFR from the Golgi compartment to the cell surface is functionally important.
“EHD1 or RUSC2 depletion impaired the EGF-induced cell proliferation, demonstrating that the novel, EHD1- and RUSC2-dependent transport of unstimulated EGFR from the Golgi compartment to the cell surface that we describe is functionally important, with implications for physiologic and oncogenic roles of EGFR and targeted cancer therapies.”
Quote in the paperSubstantial claim - Interpretationown data§ RESULTS
EHD1 regulates the exit of the EGFR pool that enters the Golgi compartment after endocytosis and retrograde transport from the cell surface.
“This result indicates that EHD1 also regulates the exit of the EGFR pool that enters the Golgi compartment after endocytosis and retrograde transport from the cell surface.”
Quote in the paperSubstantial claim- Rests on an assumption: EHD1 is currently thought to work primarily at the endosomes.
- Premisecitation§ INTRODUCTION
EHD1 and mutant EGFR colocalize in the endocytic recycling compartment in NSCLC.
“In previous work investigating the subcellular organelles where constitutively active non-small cell lung cancer (NSCLC)-associated EGFR mutants traffic, we observed the colocalization of EHD1 and mutant EGFR in the endocytic recycling compartment (42).”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [42] Chung BM, Raja SM, Clubb RJ, Tu C, George M, Band V, Band H. 2009. Aberrant trafficking of NSCLC-associated EGFR mutants through the endocytic recycling pathway promotes interaction with Src. BMC Cell Biol 10:84. doi: 10.1186/1471-2121-10-84. [DOI] [PMC free article] [PubMed] [Google Scholar] source
- Premisecitation§ INTRODUCTION
EHD1 is required for the basal surface expression of the RTK CSF1 receptor in wild-type and EHD1-null bone marrow-derived macrophages.
“Recently, using wild-type and EHD1-null bone marrow-derived macrophages, we found that EHD1 is required for the basal surface expression of the RTK colony-stimulating factor 1 (CSF1) receptor (41).”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [41] Cypher LR, Bielecki TA, Huang L, An W, Iseka F, Tom E, Storck MD, Hoppe AD, Band V, Band H. 2016. Corrigendum to CSF-1 receptor signalling is governed by pre-requisite EHD1 mediated receptor display on the macrophage cell surface [Cell Signalling 2016 Sep.; 28(9): 1325–35. Cell Signal 28:1933. doi: 10.1016/j.cellsig.2016.07.018. [DOI] [PMC free article] [PubMed] [Google Scholar] source
Checked before — its earlier verdict is held back until a new check runs.
- Study design and conductreasoning§ RESULTS
The use of multiple distinct siRNAs or shRNAs and analysis of knockout MEFs make any off-target effects of the knockdown approach remote.
“Although the use of multiple distinct siRNAs or shRNAs and analysis of knockout MEFs (see below) make any off-target effects of the knockdown approach remote, we carried out rescue analyses to rigorously validate our findings.”
Quote in the paperNot a substantial claim - Interpretationown data§ RESULTS
EHD1 plays a role in anterograde trafficking of EGFR out of the Golgi compartment.
“The Golgi retention of EGFR in EHD1-depleted cells following a switch to EGF-free medium and the results of metabolic pulse-chase analyses support a role of EHD1 in anterograde trafficking of EGFR out of the Golgi compartment.”
Quote in the paperSubstantial claim - Interpretationown data§ RESULTS
EHD1 plays a key positive role in the maintenance of total and cell surface levels of EGFR under basal, ligand-unstimulated conditions.
“Based on these findings, we conclude that EHD1 plays a key positive role in the maintenance of total and cell surface levels of EGFR under basal, ligand-unstimulated conditions.”
Quote in the paperSubstantial claim - Interpretationown data§ INTRODUCTION
EHD1 and RUSC2 function as positive regulators of the transport of unstimulated EGFR from the Golgi compartment to the cell surface to maintain an activation-ready cell surface pool of EGFR.
“Here, we show that EHD1 and RUSC2 show an overlapping subcellular localization and function as positive regulators of the transport of unstimulated EGFR from the Golgi compartment to the cell surface to help maintain an activation-ready cell surface pool of EGFR on the plasma membrane.”
Quote in the paperSubstantial claim- Rests on an assumption: Cell surface display of EGFR is essential for cellular responses to its ligands.
- Premisecitation§ INTRODUCTION
EHD1 levels in NSCLC show a negative correlation with sensitivity to EGFR-targeted kinase inhibitor therapy.
“EHD1 levels in NSCLC also showed a negative correlation with sensitivity to EGFR-targeted kinase inhibitor therapy (47).”
- Also stated in: § DISCUSSION · plain
“and EHD1 levels in NSCLC showed a negative correlation with sensitivity to EGFR-targeted kinase inhibitor therapy (47).”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [47] Wang X, Yin H, Zhang H, Hu J, Lu H, Li C, Cao M, Yan S, Cai L. 2018. NF-kappaB-driven improvement of EHD1 contributes to erlotinib resistance in EGFR-mutant lung cancers. Cell Death Dis 9:418. doi: 10.1038/s41419-018-0447-7. [DOI] [PMC free article] [PubMed] [Google Scholar] source
- Also stated in: § DISCUSSION · plain
- Interpretationown data§ RESULTS
RUSC2 and EHD1 function in a common pathway to positively regulate basal traffic of EGFR from the Golgi compartment to the cell surface to ensure optimal surface receptor levels for subsequent ligand-mediated activation and cellular responses.
“Collectively, these results support our conclusion that RUSC2 and EHD1 function in a common pathway to positively regulate the basal traffic of EGFR from the Golgi compartment to the cell surface to ensure optimal surface receptor levels for subsequent ligand-mediated activation and cellular responses.”
Quote in the paperSubstantial claim - Interpretationreasoning§ DISCUSSION
The control of endocytic as well as biosynthetic pools of a prototype RTK at the Golgi compartment is unprecedented.
“Thus, the control of endocytic as well as biosynthetic pools of a prototype RTK at the Golgi compartment is unprecedented.”
Quote in the paperSubstantial claim - Interpretationown data§ DISCUSSION
The study provides the first direct evidence for a role of RUSC2 in anterograde EGFR trafficking out of the Golgi compartment.
“Thus, our study provides the first direct evidence for a role of RUSC2 in anterograde EGFR trafficking out of the Golgi compartment and helps describe a novel biochemical duo of EHD1 and RUSC2 that regulates EGFR display on the cell surface.”
Quote in the paperSubstantial claim - Premisecitation§ RESULTS
The increase in radiolabel during the 4-h chase in control cells is consistent with EGFR becoming more antibody reactive as the receptor undergoes posttranslational modification.
“The increase in radiolabel during the 4-h chase in control cells (Fig. 5C) is consistent with the EGFR becoming more antibody reactive as the receptor undergoes posttranslational modification, as shown previously (65, 66).”
Quote in the paperNot a substantial claim0 of 2 sources checked- cites [65] Franovic A, Gunaratnam L, Smith K, Robert I, Patten D, Lee S. 2007. Translational up-regulation of the EGFR by tumor hypoxia provides a nonmutational explanation for its overexpression in human cancer. Proc Natl Acad Sci U S A 104:13092–13097. doi: 10.1073/pnas.0702387104. [DOI] [PMC free article] [PubMed] [Google Scholar] source
- cites [66] Gamou S, Shimizu N. 1987. Change in metabolic turnover is an alternate mechanism increasing cell surface epidermal growth factor receptor levels in tumor cells. J Biol Chem 262:6708–6713. [PubMed] [Google Scholar] source
- Interpretationown data§ DISCUSSION
EHD1 and RUSC2 form a novel biochemical duo that regulates EGFR display on the cell surface.
“and helps describe a novel biochemical duo of EHD1 and RUSC2 that regulates EGFR display on the cell surface.”
Quote in the paperSubstantial claim - Interpretationown data§ DISCUSSION
Reduced levels and surface display of EGFR in EHD1 knockdown cells translate into impaired signaling and cell proliferation.
“Indeed, we demonstrate that the reduced levels and surface display of EGFR in EHD1 KD cells translate into impaired signaling and cell proliferation (Fig. 6).”
Quote in the paperSubstantial claim - Interpretationown data§ RESULTS
EHD1 plays a key positive role in the cell surface expression of unstimulated EGFR.
“In view of our findings that EHD1 plays a key positive role in the cell surface expression of unstimulated EGFR, we were intrigued by a proteomics analysis of unstimulated EGFR-associated protein complex in which RUSC2 was reported as an uncharacterized component (53).”
Quote in the paperSubstantial claim - Interpretationown data§ DISCUSSION
Much of the Golgi compartment-localized EGFR represents newly synthesized protein on its way to the cell surface.
“our results indicate that much of the Golgi compartment-localized EGFR represents the newly synthesized protein on its way to the cell surface (Fig. 5C).”
Quote in the paperSubstantial claim
- It may state two separate claims as one. 25
- The quote only partly states this claim. 2
- Probably not a substantial claim. 21
It may state two separate claims as one.
Findingown data§ ABSTRACTInducible EHD1 knockdown reduces cell surface EGFR expression with accumulation at the Golgi compartment.
“Inducible EHD1 knockdown reduced the cell surface EGFR expression with accumulation at the Golgi compartment, a phenotype rescued by exogenous EHD1.”
Quote in the paperSubstantial claimIt may state two separate claims as one.
Findingown data§ RESULTSCells coexpressing shRNA-resistant EHD1-GFP with EHD1-specific shRNA display total EGFR levels comparable to EHD1-GFP-expressing cells with control shRNA, whereas cells with control empty vector and EHD1 shRNA display reduced EGFR levels.
“Cells coexpressing the shRNA-resistant EHD1-GFP together with EHD1-specific shRNA displayed total EGFR levels comparable to those in the EHD1-GFP-expressing cell line coexpressing a control shRNA. In contrast, cells transduced with only a control empty vector in conjunction with the EHD1 shRNA displayed reduced EGFR levels (Fig. 1I and J).”
Quote in the paperSubstantial claimIt may state two separate claims as one.
Findingown data§ RESULTSEHD1 knockdown cells without EHD1 rescue exhibit strong perinuclear EGFR accumulation with correspondingly lower surface EGFR signals.
“In contrast, cells with EHD1 KD but without EHD1 rescue exhibited strong perinuclear EGFR accumulation with correspondingly lower surface EGFR signals (Fig. 5A).”
Quote in the paperSubstantial claimIt may state two separate claims as one.
Findingown data§ RESULTSFull-length RUSC2 does not bind the EHD1 EH domain, and no co-immunoprecipitation of ectopically cotransfected EHD1 and RUSC2 is detected.
“However, we could not detect the binding of full-length RUSC2 with the EHD1 EH domain, nor could we detect co-IP of ectopically cotransfected EHD1 and RUSC2, either when immunoprecipitated hemagglutinin (HA)-RUSC2 was incubated with lysates of cells transfected with Myc-EHD1 or when HA-RUSC2 and Myc-EHD1 were cotransfected in HEK-293T cells (Fig. 7B and C).”
- Also stated in: § DISCUSSION · plain
“While RUSC2 did not show a physical interaction with EHD1 in cells (Fig. 7B and C)”
Quote in the paperSubstantial claim- Also stated in: § DISCUSSION · plain
The quote only partly states this claim.
Findingown data§ RESULTSEHD1 knockdown reduces EGF-dependent cell proliferation in 16A5 cells.
“Both EHD1 KD 16A5 lines displayed significantly reduced proliferation relative to that of control shRNA-expressing cells (Fig. 6C).”
Quote in the paperSubstantial claimIt may state two separate claims as one.
Findingown data§ RESULTSExogenous EHD1-DsRed colocalizes with endogenous EGFR in 16A5 cells even without monensin treatment, and more robustly in monensin-treated cells.
“When an exogenous EHD1-DsRed construct was expressed via transient transfection, the colocalization with endogenous EGFR became obvious even in the absence of monensin treatment (Fig. 4C) and was more robust in monensin-treated cells (Fig. 4D).”
Quote in the paperSubstantial claimIt may state two separate claims as one.
Findingown data§ RESULTSEHD1 and EGFR do not physically interact, as no co-immunoprecipitation between them was observed.
“No co-IP between EHD1 and EGFR, however, was observed.”
Quote in the paperSubstantial claimThe quote only partly states this claim.
Findingown data§ RESULTSEHD1 knockdown moderately reduces EGFR accumulation after serum and EGF deprivation in S2013 cells.
“or a pancreatic adenocarcinoma cell line, S2013 (Fig. 3E).”
Quote in the paperSubstantial claimIt may state two separate claims as one.
Findingown data§ RESULTSIn EHD1 knockdown 16A5 cells, a smaller fraction of newly synthesized EGFR is converted to the mature form and with a delay.
“a smaller fraction of the newly synthesized EGFR was converted to the mature form in EHD1 KD 16A5 cells and with a delay (Fig. 5C).”
Quote in the paperSubstantial claimIt may state two separate claims as one.
Findingown data§ RESULTSRUSC2 knockdown using two independent shRNAs substantially impairs accumulation of total EGFR protein upon EGF withdrawal compared to control cells, and this effect is more robust than that seen with EHD1 knockdown.
“RUSC2 KD, using two independent shRNAs, led to a substantial impairment of the accumulation of total EGFR protein upon EGF withdrawal compared to levels in control cells; this effect was even more robust than that seen with EHD1 KD (Fig. 8A and B).”
Quote in the paperSubstantial claimIt may state two separate claims as one.
Findingown data§ RESULTSRUSC2 knockdown significantly decreases cell surface EGFR levels compared to control cells, mimicking the impact of EHD1 knockdown.
“Flow cytometry analyses of cells treated with Dox for 48 h in EGF-containing medium followed by Dox in serum and EGF deprivation medium for 24 h revealed a significant decrease in the cell surface EGFR levels in RUSC2 KD cells compared to levels in control cells, mimicking the impact of EHD1 KD (Fig. 8C and D).”
Quote in the paperSubstantial claimIt may state two separate claims as one.
Findingown data§ DISCUSSIONEHD1 depletion leads to accumulation of EGFR in the Golgi compartment with concurrent reduction in trafficking of EGFR to the cell surface.
“We demonstrate that EHD1 depletion leads to accumulation of EGFR in this compartment, with concurrent reduction in the trafficking of EGFR to the cell surface (Fig. 5A).”
Quote in the paperSubstantial claimProbably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.
Premisecitation§ INTRODUCTIONEHD1 and EGFR expression levels are increased in human papillary thyroid cancer, and EHD1 protein levels are significantly associated with tumor size, lymph node metastasis, and EGFR expression.
“EHD1 and EGFR expression levels were also reported to be increased in human papillary thyroid cancer, and levels of EHD1 protein were significantly associated with tumor size, lymph node metastasis, and EGFR expression (46).”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [46] Liu Y, Liang Y, Li M, Liu D, Tang J, Yang W, Tong D, Jin X. 2018. Eps15 homology domain 1 promotes the evolution of papillary thyroid cancer by regulating endocytotic recycling of epidermal growth factor receptor. Oncol Lett 16:4263–4270. doi: 10.3892/ol.2018.9200. [DOI] [PMC free article] [PubMed] [Google Scholar] source
It may state two separate claims as one.
Study design and conductown data§ RESULTSMonensin treatment increases accumulation of EGFR in perinuclear vesicles in 16A5 cells, indicating effective inhibition of cargo exit from endocytic compartments.
“monensin treatment of 16A5 cells increased the accumulation of EGFR in perinuclear vesicles, indicating an effective inhibition of cargo exit from endocytic compartments.”
Quote in the paperNot a substantial claimProbably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.
Premisecitation§ INTRODUCTIONIn breast cancer, EHD1-positive patients have shorter overall and disease-free survival, and EHD1-positive plus phospho-EGFR-positive status is even more predictive of shorter survival.
“EHD1-positive (EHD1+) patients had a shorter overall and disease-free survival, and a patient status of EHD1+ plus phospho-EGFR+ was even more predictive of shorter survival (45).”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [45] Tong D, Liang YN, Stepanova AA, Liu Y, Li X, Wang L, Zhang F, Vasilyeva NV. 2017. Increased Eps15 homology domain 1 and RAB11FIP3 expression regulate breast cancer progression via promoting epithelial growth factor receptor recycling. Tumour Biol 39:1010428317691010. doi: 10.1177/1010428317691010. [DOI] [PubMed] [Google Scholar] source
Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.
Premisecitation§ INTRODUCTIONIn breast cancer, EHD1 and RAB11-FIP3 expression positively correlate, and these markers correlate with phospho-EGFR levels in tumors.
“A similar analysis in breast cancer showed a positive correlation between EHD1 and RAB11-FIP3 expression, and the expression of these markers correlated with phospho-EGFR levels in tumors;”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [45] Tong D, Liang YN, Stepanova AA, Liu Y, Li X, Wang L, Zhang F, Vasilyeva NV. 2017. Increased Eps15 homology domain 1 and RAB11FIP3 expression regulate breast cancer progression via promoting epithelial growth factor receptor recycling. Tumour Biol 39:1010428317691010. doi: 10.1177/1010428317691010. [DOI] [PubMed] [Google Scholar] source
Probably not a substantial claim. The paper’s conclusions may not depend on it.
Premisecitation§ INTRODUCTIONDoxycycline-inducible EHD3 expression in EHD3-deleted glioblastoma cell lines increases basal levels of EGFR.
“although doxycycline (Dox)-inducible EHD3 expression in EHD3-deleted glioblastoma cell lines increased the basal levels of EGFR (48).”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [48] Amessou M, Ebrahim AS, Dilly A, Joseph M, Tabolina M, Chukkapalli S, Meroueh L, Syed JT, Liddane A, Lang SL, Al-Katib A, Kandouz M. 2016. Spatio-temporal regulation of EGFR signaling by the Eps15 homology domain-containing protein 3 (EHD3). Oncotarget 7:79203–79216. doi: 10.18632/oncotarget.13008. [DOI] [PMC free article] [PubMed] [Google Scholar] source
Probably not a substantial claim. The paper’s conclusions may not depend on it.
Premisecitation§ RESULTSEHD1 is required for exit of MHC-I from the endocytic recycling compartment back to the cell surface.
“Previous studies in which MHC-I internalized from the cell surface was tracked showed that EHD1 is required for exit of MHC-I from the endocytic recycling compartment back to the cell surface (Fig. 2C and D).”
Quote in the paperNot a substantial claimProbably not a substantial claim. The paper’s conclusions may not depend on it.
Premisecitation§ INTRODUCTIONRUSC2 was identified in an EGFR-associated protein complex isolated from cells in the absence of ligand stimulation.
“We noted RUSC2 as an uncharacterized protein identified in an EGFR-associated protein complex isolated from cells in the absence of ligand stimulation (53).”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [53] Deribe YL, Wild P, Chandrashaker A, Curak J, Schmidt MHH, Kalaidzidis Y, Milutinovic N, Kratchmarova I, Buerkle L, Fetchko MJ, Schmidt P, Kittanakom S, Brown KR, Jurisica I, Blagoev B, Zerial M, Stagljar I, Dikic I. 2009. Regulation of epidermal growth factor receptor trafficking by lysine deacetylase HDAC6. Sci Signal 2:ra84. doi: 10.1126/scisignal.2000576. [DOI] [PubMed] [Google Scholar] source
Probably not a substantial claim.
Premisecitation§ AbstractPostactivation endocytic trafficking of EGFR has been well elucidated.
“While postactivation endocytic trafficking of EGFR has been well elucidated, little is known about mechanisms of basal/preactivation surface display of EGFR.”
- Also stated in: § INTRODUCTION · boosted
“Given key physiological and oncogenic roles of EGFR, many previous investigations have elucidated mechanisms that control the endocytic trafficking of ligand-activated EGFR, including the ligand-induced activation of both clathrin-dependent and clathrin-independent endocytosis and differential transport into lysosomal degradation or endocytic recycling based on the nature of activating ligands and”
Quote in the paperSubstantial claim0 of 2 sources checked- cites [10] Sigismund S, Woelk T, Puri C, Maspero E, Tacchetti C, Transidico P, Di Fiore PP, Polo S. 2005. Clathrin-independent endocytosis of ubiquitinated cargos. Proc Natl Acad Sci U S A 102:2760–2765. doi: 10.1073/pnas.0409817102. [DOI] [PMC free article] [PubMed] [Google Scholar] source
- cites [11] Roepstorff K, Grandal MV, Henriksen L, Knudsen SL, Lerdrup M, Grovdal L, Willumsen BM, van Deurs B. 2009. Differential effects of EGFR ligands on endocytic sorting of the receptor. Traffic 10:1115–1127. doi: 10.1111/j.1600-0854.2009.00943.x. [DOI] [PMC free article] [PubMed] [Google Scholar] source
- Also stated in: § INTRODUCTION · boosted
Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.
Premisecitation§ RESULTSMEFs express EHD1 and other EHD family members, and EHD1 is critical for primary cilium biogenesis in these cells.
“mouse embryonic fibroblasts (MEFs), the other cell type we use, are known to express EHD1 and other family members as well, and EHD1 was critical for primary cilium biogenesis in these cells (61).”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [61] Bhattacharyya S, Rainey MA, Arya P, Mohapatra BC, Mushtaq I, Dutta S, George M, Storck MD, McComb RD, Muirhead D, Todd GL, Gould K, Datta K, Gelineau-van Waes J, Band V, Band H. 2016. Endocytic recycling protein EHD1 regulates primary cilia morphogenesis and SHH signaling during neural tube development. Sci Rep 6:20727. doi: 10.1038/srep20727. [DOI] [PMC free article] [PubMed] [Google Scholar] source
Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.
Premisecitation§ AbstractEGFR is a prototype receptor tyrosine kinase and an oncoprotein in many solid tumors.
“Epidermal growth factor receptor (EGFR) is a prototype receptor tyrosine kinase and an oncoprotein in many solid tumors.”
- Also stated in: § INTRODUCTION · plain
“Activation of ErbB receptors is linked to the initiation and progression of human cancers, and the levels of their expression are often significantly elevated in many solid tumors (e.g., breast, colon, ovary, pancreas, and glial) (6–8).”
Quote in the paperNot a substantial claim0 of 3 sources checked- cites [6] Rowinsky EK. 2004. The ErbB family: targets for therapeutic development against cancer and therapeutic strategies using monoclonal antibodies and tyrosine kinase inhibitors. Annu Rev Med 55:433–457. doi: 10.1146/annurev.med.55.091902.104433. [DOI] [PubMed] [Google Scholar] source
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- cites [8] Santarius T, Shipley J, Brewer D, Stratton MR, Cooper CS. 2010. A census of amplified and overexpressed human cancer genes. Nat Rev Cancer 10:59–64. doi: 10.1038/nrc2771. [DOI] [PubMed] [Google Scholar] source
- Also stated in: § INTRODUCTION · plain
It may state two separate claims as one.
Study design and conductown data§ RESULTSThe exogenous EHD1-GFP fusion construct lacks the 3′ UTR of EHD1 and is resistant to the EHD1 3′ UTR-targeted shRNAs.
“We generated 16A5 cell lines that coexpress an exogenous Dox-inducible EHD1-green fluorescent protein (GFP) fusion construct lacking the 3′ UTR of EHD1, and thus resistant to the EHD1 3′ UTR-targeted shRNAs, together with the EHD1-directed shRNA or a control shRNA.”
Quote in the paperSubstantial claimIt may state two separate claims as one.
Study design and conductcitation§ RESULTS16A5 is a nontumorigenic, HPV16 E6/E7-immortalized, EGF-dependent human mammary epithelial cell line that displays expected ligand-induced EGFR degradation kinetics.
“16A5 is a nontumorigenic, human papillomavirus 16 (HPV16) E6/E7-immortalized and EGF-dependent human mammary epithelial cell (MEC) line (55) that displays the expected ligand-induced EGFR degradation kinetics (56–60) (Fig. 1A).”
Quote in the paperSubstantial claim0 of 6 sources checked- cites [55] Band V, Sager R. 1989. Distinctive traits of normal and tumor-derived human mammary epithelial cells expressed in a medium that supports long-term growth of both cell types. Proc Natl Acad Sci U S A 86:1249–1253. doi: 10.1073/pnas.86.4.1249. [DOI] [PMC free article] [PubMed] [Google Scholar] source
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Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.
Premisecitation§ INTRODUCTIONRUSC2 interacts with the Golgi protein GM130 and has been proposed as a potential regulator of endosomal targeting of vesicles derived from the endoplasmic reticulum.
“RUSC2 has also been reported to interact with the Golgi protein GM130 and has been proposed as a potential regulator of endosomal targeting of vesicles derived from the endoplasmic reticulum (ER) (50).”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [50] Bayer M, Fischer J, Kremerskothen J, Ossendorf E, Matanis T, Konczal M, Weide T, Barnekow A. 2005. Identification and characterization of Iporin as a novel interaction partner for Rab1. BMC Cell Biol 6:15. doi: 10.1186/1471-2121-6-15. [DOI] [PMC free article] [PubMed] [Google Scholar] source
Probably not a substantial claim. The authors may not put it forward as true. The paper’s conclusions may not depend on it.
Premisecitation§ INTRODUCTIONEHD3 may function in targeting ligand-stimulated EGFR to lysosomal degradation by diverting it from endocytic recycling.
“A recent study suggested that EHD3 may function in targeting the ligand-stimulated EGFR to lysosomal degradation by diverting it from endocytic recycling (48), consistent with EHD3 deletion in glioblastoma (49), although doxycycline (Dox)-inducible EHD3 expression in EHD3-deleted glioblastoma cell lines increased the basal levels of EGFR (48).”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [48] Amessou M, Ebrahim AS, Dilly A, Joseph M, Tabolina M, Chukkapalli S, Meroueh L, Syed JT, Liddane A, Lang SL, Al-Katib A, Kandouz M. 2016. Spatio-temporal regulation of EGFR signaling by the Eps15 homology domain-containing protein 3 (EHD3). Oncotarget 7:79203–79216. doi: 10.18632/oncotarget.13008. [DOI] [PMC free article] [PubMed] [Google Scholar] source
Probably not a substantial claim. The paper’s conclusions may not depend on it.
Premisecitation§ INTRODUCTIONRUSC2 contains a RUN domain that interacts with Rab1 and Rab35 small GTPases.
“RUSC2/iporin contains a RUN domain (named based on its presence in RPIP8, UNC-14, and NESCA proteins) that interacts with Rab1 and Rab35 small GTPases (51, 52).”
Quote in the paperNot a substantial claim0 of 2 sources checked- cites [51] Chi S, Cao H, Wang Y, McNiven MA. 2011. Recycling of the epidermal growth factor receptor is mediated by a novel form of the clathrin adaptor protein Eps15. J Biol Chem 286:35196–35208. doi: 10.1074/jbc.M111.247577. [DOI] [PMC free article] [PubMed] [Google Scholar] source
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It may state two separate claims as one.
Interpretationown data§ DISCUSSIONEHD1 and RUSC2 function in a common trafficking pathway but independent of an interaction.
“suggesting that they function in a common trafficking pathway but independent of an interaction.”
Quote in the paperSubstantial claimIt may state two separate claims as one.
Interpretationown data§ DISCUSSIONUnstimulated EGFR transported through both anterograde and retrograde mechanisms reaches an EHD1-dependent Golgi compartment, and EHD1 is required for EGFR to be recycled to the cell surface from this compartment.
“it is likely that unstimulated EGFR transported through both anterograde and retrograde mechanisms reaches an EHD1-dependent Golgi compartment and that EHD1 is required for EGFR to be recycled to the cell surface from this compartment.”
Quote in the paperSubstantial claimProbably not a substantial claim. It may state two separate claims as one.
Premisecitation§ RESULTSRUSC2 directly interacts with GM130 and with Golgi-associated Rab1.
“Given the previous reports that RUSC2 directly interacts with GM130 as well as with Golgi-associated Rab1 (50), the prominent colocalization of RUSC2 with EHD1 led us to hypothesize that RUSC2, similar to EHD1, may function to promote the EGFR trafficking out of the Golgi compartment.”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [50] Bayer M, Fischer J, Kremerskothen J, Ossendorf E, Matanis T, Konczal M, Weide T, Barnekow A. 2005. Identification and characterization of Iporin as a novel interaction partner for Rab1. BMC Cell Biol 6:15. doi: 10.1186/1471-2121-6-15. [DOI] [PMC free article] [PubMed] [Google Scholar] source
Probably not a substantial claim. The paper’s conclusions may not depend on it.
Study design and conductown data§ MATERIALS AND METHODSFACS analyses of cell surface EGFR and MHC-I used five replicates per condition.
“16A5 cells were seeded at 105 cells per well of six-well plates (five replicates per condition) and grown in regular medium in the presence of Dox for 48 h.”
Quote in the paperNot a substantial claimProbably not a substantial claim.
Study design and conductown data§ MATERIALS AND METHODSThe anti-RUSC2 antibody was validated using epitope-tagged RUSC2 expressed in 293T cells and shRNA knockdown of endogenous RUSC2.
“A rabbit polyclonal antibody against human RUSC2/iporin was generated against an internal peptide sequence (amino acid residues 532 to 553) through a commercial vendor (Pacific Immunology, Ramona, CA) and validated using epitope-tagged RUSC2 expressed in 293T cells and shRNA knockdown of endogenous RUSC2.”
Quote in the paperSubstantial claimIt may state two separate claims as one.
Interpretationown data§ DISCUSSIONStudies presented here demonstrate a new and functionally important role of EHD1 and a potential novel partner, RUSC2, in the control of Golgi compartment-to-surface transport of a prototype RTK, EGFR, under ligand-unstimulated conditions.
“In conclusion, studies presented here demonstrate a new and functionally important role of EHD1 and a potential novel partner, RUSC2, in the control of Golgi compartment-to-surface transport of a prototype RTK, EGFR under ligand-unstimulated conditions.”
Quote in the paperSubstantial claimIt may state two separate claims as one.
Premisecitation§ RESULTSPrevious studies of nuclear localization of EGFR concluded that EGFR traffics via a retrograde endocytic route, but such studies were done in the presence of ligands.
“Previous studies of nuclear localization of EGFR have concluded that EGFR traffics via a retrograde endocytic route, but such studies were done in the presence of ligands (67).”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [67] Wang YN, Wang H, Yamaguchi H, Lee HJ, Lee HH, Hung MC. 2010. COPI-mediated retrograde trafficking from the Golgi to the ER regulates EGFR nuclear transport. Biochem Biophys Res Commun 399:498–504. doi: 10.1016/j.bbrc.2010.07.096. [DOI] [PMC free article] [PubMed] [Google Scholar] source
Probably not a substantial claim.
Study design and conductown data§ MATERIALS AND METHODSA tightly inducible Tet-on clone was used for stable transfection with pRevTRE-EHD1-GFP or the empty vector.
“A tightly inducible clone was used for stable transfection with pRevTRE-EHD1-GFP or the empty vector (pRevTRE-GFP) using Lipofectamine 2000 and selected in 50 μg/ml hygromycin, and clones were analyzed for inducible EHD1-GFP or GFP (vector only clones) after 7-day culture with 1 μg/ml Dox using Western blot analysis.”
Quote in the paperSubstantial claimProbably not a substantial claim. The paper’s conclusions may not depend on it.
Premisecitation§ DISCUSSIONEndocytic recycling of MHC-I, tracked using antibody binding at the cell surface, is impaired upon EHD1 knockdown.
“since endocytic recycling of MHC-I, tracked using antibody binding at the cell surface, is known to be impaired upon EHD1 KD (23).”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [23] Caplan S, Naslavsky N, Hartnell LM, Lodge R, Polishchuk RS, Donaldson JG, Bonifacino JS. 2002. A tubular EHD1-containing compartment involved in the recycling of major histocompatibility complex class I molecules to the plasma membrane. EMBO J 21:2557–2567. doi: 10.1093/emboj/21.11.2557. [DOI] [PMC free article] [PubMed] [Google Scholar] source
Probably not a substantial claim. The paper’s conclusions may not depend on it.
Premisecitation§ DISCUSSIONEHD1 plays a key role in the surface expression of CSF1R on primary macrophages.
“Consistent with a broader role of EHD1 in RTK trafficking, we have recently found that EHD1 also plays a key role in the surface expression of the CSF1R, an RTK, on primary macrophages (41).”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [41] Cypher LR, Bielecki TA, Huang L, An W, Iseka F, Tom E, Storck MD, Hoppe AD, Band V, Band H. 2016. Corrigendum to CSF-1 receptor signalling is governed by pre-requisite EHD1 mediated receptor display on the macrophage cell surface [Cell Signalling 2016 Sep.; 28(9): 1325–35. Cell Signal 28:1933. doi: 10.1016/j.cellsig.2016.07.018. [DOI] [PMC free article] [PubMed] [Google Scholar] source
Checked before — its earlier verdict is held back until a new check runs.
Probably not a substantial claim. The paper’s conclusions may not depend on it.
Premisecitation§ DISCUSSIONEHD1 knockdown impairs biosynthetic transport of a VSVG-GFP fusion protein used as a marker of transport from the Golgi compartment to the cell surface.
“It is notable that EHD1 knockdown was shown to impair the biosynthetic transport of a vesicular stomatitis virus G protein (VSVG)-GFP fusion protein used as a marker of the transport from the Golgi compartment to the cell surface (73), but no further studies along these lines have been reported.”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [73] Zhang J, Reiling C, Reinecke JB, Prislan I, Marky LA, Sorgen PL, Naslavsky N, Caplan S. 2012. Rabankyrin-5 interacts with EHD1 and Vps26 to regulate endocytic trafficking and retromer function. Traffic 13:745–757. doi: 10.1111/j.1600-0854.2012.01334.x. [DOI] [PMC free article] [PubMed] [Google Scholar] source
It may state two separate claims as one.
Premisecitation§ DISCUSSIONEHD1 expression is associated with poor prognosis in non-small cell lung cancer and breast cancer.
“EHD1 expression was recently found to be associated with poor prognosis in non-small cell lung cancer (43, 44) and breast cancer (45), and EHD1 levels in NSCLC showed a negative correlation with sensitivity to EGFR-targeted kinase inhibitor therapy (47).”
Quote in the paperNot a substantial claim0 of 3 sources checked- cites [43] Lu H, Meng Q, Wen Y, Hu J, Zhao Y, Cai L. 2013. Increased EHD1 in non-small cell lung cancer predicts poor survival. Thorac Cancer 4:422–432. doi: 10.1111/1759-7714.12043. [DOI] [PubMed] [Google Scholar]
- cites [44] Gao Y, Wang Y, Sun L, Meng Q, Cai L, Dong X. 2014. Expression of TGFbeta-1 and EHD1 correlated with survival of non-small cell lung cancer. Tumour Biol 35:9371–9380. doi: 10.1007/s13277-014-2164-x. [DOI] [PubMed] [Google Scholar] source
- cites [45] Tong D, Liang YN, Stepanova AA, Liu Y, Li X, Wang L, Zhang F, Vasilyeva NV. 2017. Increased Eps15 homology domain 1 and RAB11FIP3 expression regulate breast cancer progression via promoting epithelial growth factor receptor recycling. Tumour Biol 39:1010428317691010. doi: 10.1177/1010428317691010. [DOI] [PubMed] [Google Scholar] source
Probably not a substantial claim.
Premisecitation§ RESULTSRUSC2-interacting small G-protein Rab1a regulates trafficking from the Golgi compartment to the plasma membrane.
“Given the above results, the reported RUSC2 interaction with GM130 (50) and the role of RUSC2-interacting small G-protein Rab1a in regulating trafficking from the Golgi compartment to the plasma membrane (50, 69, 70), we reasoned that RUSC2 may also function in EHD1-dependent EGFR trafficking out of the Golgi compartment.”
Quote in the paperSubstantial claim0 of 3 sources checked- cites [50] Bayer M, Fischer J, Kremerskothen J, Ossendorf E, Matanis T, Konczal M, Weide T, Barnekow A. 2005. Identification and characterization of Iporin as a novel interaction partner for Rab1. BMC Cell Biol 6:15. doi: 10.1186/1471-2121-6-15. [DOI] [PMC free article] [PubMed] [Google Scholar] source
- cites [69] Allan BB, Moyer BD, Balch WE. 2000. Rab1 recruitment of p115 into a cis-SNARE complex: programming budding COPII vesicles for fusion. Science 289:444–448. doi: 10.1126/science.289.5478.444. [DOI] [PubMed] [Google Scholar] source
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Probably not a substantial claim.
Premisecitation§ RESULTSRUSC2 was reported as an uncharacterized component in a proteomics analysis of an unstimulated EGFR-associated protein complex.
“we were intrigued by a proteomics analysis of unstimulated EGFR-associated protein complex in which RUSC2 was reported as an uncharacterized component (53).”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [53] Deribe YL, Wild P, Chandrashaker A, Curak J, Schmidt MHH, Kalaidzidis Y, Milutinovic N, Kratchmarova I, Buerkle L, Fetchko MJ, Schmidt P, Kittanakom S, Brown KR, Jurisica I, Blagoev B, Zerial M, Stagljar I, Dikic I. 2009. Regulation of epidermal growth factor receptor trafficking by lysine deacetylase HDAC6. Sci Signal 2:ra84. doi: 10.1126/scisignal.2000576. [DOI] [PubMed] [Google Scholar] source
The paper relies on this without support.
Cell surface display of EGFR is essential for cellular responses to its ligands.
“Cell surface display of EGFR is essential for cellular responses to its ligands.”
The paper relies on this without support.
Little is known about mechanisms of basal/preactivation surface display of EGFR.
“While postactivation endocytic trafficking of EGFR has been well elucidated, little is known about mechanisms of basal/preactivation surface display of EGFR.”
The paper relies on this without support.
How EHD1 is involved in regulating EGFR biology is not known.
“How EHD1 may be involved in regulating EGFR biology, however, is not known.”
The paper relies on this without support.
EHD1 is currently thought to work primarily at the endosomes.
“Since EHD1 is currently thought to work primarily at the endosomes, we tested and found support for the idea that a portion of the Golgi compartment-localized EGFR is derived from the pool internalized from the cell surface (Fig. 5A).”
Self-check
Percentages, sums and reported differences re-computed from the paper’s own numbers.
Each contradiction between two passages listed here was confirmed by an independent check.
Cell-surface tracing result cites the wrong figure panel
MinorThe discussion cites Fig. 5A as support for EGFR arriving from the cell surface, but the results identify the cell-surface-prelabeling experiment and its finding as Fig. 5D.
“we tested and found support for the idea that a portion of the Golgi compartment-localized EGFR is derived from the pool internalized from the cell surface (Fig. 5A).”
“Under these conditions, we observed that EGFR prelabeled at the cell surface accumulated in a GM130/Rab11 double-positive, perinuclear compartment in EHD1 KD cells, as also seen in cells that were not prelabeled on the cell surface (Fig. 5D).”
Four-hour chase conflicts with the stated 270-minute time point
MinorThe longest stated chase was 270 minutes, or 4.5 hours, rather than 4 hours.
“The increase in radiolabel during the 4-h chase in control cells (Fig. 5C)”
“a 30-, 90- or 270-min chase time before lysis”
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Metadata
- Authors
- Tom, E. C., Mushtaq, I., Mohapatra, B. C., Luan, H., Bhat, A. M., Zutshi, N., Chakraborty, S., Islam, N., Arya, P., Bielecki, T. A., Iseka, F. M., Bhattacharyya, S., Cypher, L. R., Goetz, B. T., Negi, S. K., Storck, M. D., Rana, S., Barnekow, A., Singh, P. K., Ying, G., Guda, C., Natarajan, A., Band, V., & Band, H.
- Affiliations
- University of Nebraska Medical Center (US)
- University of Münster (DE)
- Fred and Pamela Buffett Cancer Center (US)
- Tianjin Medical University Cancer Institute and Hospital (CN)
- Corresponding author
- Band, Vimla
- Band, Hamid
- ORCID
- Venue
- Molecular and Cellular Biology
- Publisher
- Informa UK Limited
- Published
- 2020-03-16
- Volume
- 40
- Issue
- 7
- ISSN
- 0270-7306 (ISSN-L)
- 1098-5549
- PMC
- PMC7076251
- OpenAlex
- W2999295226
- Cited by
- 13
- References
- 92
- Citations by year
- 2025: 2 · 2024: 4 · 2023: 2 · 2022: 3 · 2020: 2
- FWCI
- 0.62
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Locations
- Molecular and Cellular Biology (publishedVersion)
- PubMed Central (publishedVersion)
- PubMed (publishedVersion)
- Funders
- HHS | National Institutes of Health (CA87986, CA105489, CA96844, CA144027, P30 GM106397)
- U.S. Department of Defense (W81XWH-11-1-0171, W81XWH-17-1-061, W81XWH-07-1-0351)
- Nebraska Department of Health and Human Services (LB506)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Cellular transport and secretion
- Health Sciences › Medicine › Oncology › HER2/EGFR in Cancer Research
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Caveolin-1 and cellular processes
- Keywords
- epidermal growth factor receptor, EHD1, Golgi apparatus, receptor recycling, endosomal trafficking, receptor tyrosine kinase
- MeSH terms
- Animals, Carrier Proteins, Cell Communication, Cell Line, Cell Membrane, Golgi Apparatus, Humans, Protein Transport, Vesicular Transport Proteins, RNA Interference, RNA, Small Interfering, Cell Proliferation, Mice, ErbB Receptors
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Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.
A language model drew the claims from the paper on 2026-11-10, under version 3 of the claim definition (model deepseek-v4.1-flash, prompt extract-v3).
A second model labelled each claim on 2026-11-10 (model jev-1.13-20260917, definition revision gold-guideline-v0.2).
Statistics and arithmetic are recomputed, not judged.
Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.
What counts as a claim ›How we measure confidence ›How we check this ›
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