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Adaptor Protein ARH Is Recruited to the Plasma Membrane by Low Density Lipoprotein (LDL) Binding and Modulates Endocytosis of the LDL/LDL Receptor Complex in Hepatocytes
Open accessAbstract
ARH is a newly discovered adaptor protein required for the efficient activity of low density lipoprotein receptor (LDLR) in selected tissues. Individuals lacking ARH have severe hypercholesterolemia due to an impaired hepatic clearance of LDL. It has been demonstrated that ARH is required for the efficient internalization of the LDL-LDLR complex and to stabilize the association of the receptor with LDL in Epstein-Barr virus-immortalized B lymphocytes. However, little information is available on the role of ARH in liver cells. Here we provide evidence that ARH is codistributed with LDLR on the basolateral area in confluent HepG2-polarized cells. This distribution is not modified by the overexpression of LDLR. Conversely, the activation of the LDLR-mediated endocytosis, but not the binding of LDL to LDLR, promotes a significant colocalization of ARH with LDL-LDLR complex that peaked at 2 min at 37 degrees C. To further assess the role of ARH in LDL-LDLR complex internalization, we depleted ARH protein using the RNA interference technique. Twenty-four hours after transfection with ARH-specific RNA interference, ARH protein was depleted in HepG2 cells by more than 70%. Quantitative immunofluorescence analysis revealed that the depletion of ARH caused about 80% reduction in LDL internalization. Moreover, our findings indicate that ARH is associated with other proteins of the endocytic machinery. We suggest that ARH is an endocytic sorting adaptor that actively participates in the internalization of the LDL-LDLR complex, possibly enhancing the efficiency of its packaging into the endocytic vesicles.
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Written from the paper’s full text.
TL;DR
In HepG2 hepatocytes, ARH is not constitutively associated with LDLR; it is recruited to the plasma membrane and early endocytic compartments after LDL-LDLR internalization is activated, peaking at 2 min at 37 °C. ARH depletion by siRNA (>70%) blocks DiI-LDL internalization (~80% reduction), supporting ARH as an endocytic sorting adaptor for hepatic LDLR.
Key findings
- In confluent polarized HepG2 cells, ARH codistributed with LDLR in the basolateral area; this distribution was not modified by LDLR up-regulation via serum starvation.
- Activation of LDLR-mediated endocytosis, rather than LDL binding alone, promoted ARH colocalization with the LDL-LDLR complex at the plasma membrane, maximal at 2 min at 37 °C and decreasing by 5–10 min.
- ARH siRNA reduced ARH protein by ~70–80% at 24 h and caused ~80% reduction in LDL internalization; in microinjected cells, DiI-LDL internalization was blocked in 80% of siRNA-injected cells.
- ARH co-immunoprecipitated with clathrin after 2–5 min of LDL treatment and with β-adaptin, Dab2, and Rab-4 at baseline and after LDL addition; sucrose gradients showed ARH shifting toward LDLR-containing higher-density fractions 2 min after LDL.
Methods
- Human HepG2 hepatoma cells were grown to confluent polarized monolayers with bile canaliculus-like structures; LDLR endocytosis was stimulated with DiI-LDL or anti-LDLR C7 antibody under serum starvation and temperature protocols (4 °C binding followed by 37 °C warming).
- ARH was silenced with siRNA targeting ARH nucleotides 88–112, delivered by transfection or microinjection; effects were assessed by Western blotting, immunofluorescence/confocal microscopy, sucrose gradient fractionation, and co-immunoprecipitation.
- Functional LDLR assays measured DiI-LDL internalization; quantitative analysis counted cells with internalized DiI-LDL in five randomly taken areas per slide from three experiments.
Limitations
- Stated: the authors could not detect kinetics in ARH associations with β-adaptin, Dab2, and Rab-4, possibly due to limited sensitivity of semiquantitative Western blotting or difficulty establishing stoichiometry; they suggest stable transfection of HepG2 with anti-ARH siRNA might clarify this.
- Stated/evident: ARH silencing was transient, with ARH levels recovering at 48–72 h, possibly because of growth of poorly or non-transfected cells.
- Evident: findings are in the HepG2 hepatoma cell line rather than primary human hepatocytes, and the authors note their results contrast with a report that cultured arh−/− hepatocytes showed normal LDL uptake, leaving cell-type and in vivo context uncertain.
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Claims the paper makes
4 of 46 cited sources checked · 1 supported · 0 flagged · accuracy not yet measured
- Premisecitation§ Adaptor Protein ARH Is Recruited to the Plasma Membrane by Low Density Lipoprotein (LDL) Binding and Modulates Endocytosis of the LDL/LDL Receptor Complex in Hepatocytes *
In vivo studies demonstrated that ARH patients show a markedly reduced hepatic uptake of LDL similar to that found in homozygous FH.
“In vivo studies demonstrated that ARH patients show a markedly reduced hepatic uptake of LDL similar to that found in homozygous FH (10).”
Quote in the paperSubstantial claim1 of 1 sources checked- cites [10] Zuliani, 1999, Arterioscler. Thromb. Vasc. Biol. source
The available copy contains an IRIS catalog entry identifying the article (pp. 802–809) and listing “LDL turnover” as a keyword, but no study results. Searches for hepatic LDL uptake, in vivo findings, and comparison with homozygous FH found no relevant passage, so the claim cannot be checked against this copy. (LLM, conf=0.76)
Experimental — accuracy not yet measured
- Interpretationown data
ARH is not constitutively associated with the LDLR-clathrin complex in hepatocytes.
“clearly indicating that ARH is not constitutively associated with the LDLR-clathrin complex (15).”
Quote in the paperSubstantial claim1 of 1 sources checked- cites [15] Jones, 2003, J. Biol. Chem. source
- Premisecitation
ARH is required for efficient endocytosis of LDLR in circulating lymphocytes and monocyte-derived macrophages from ARH patients.
“The results of these studies clearly suggested that ARH is required for the efficient endocytosis of LDLR in these cells.”
Quote in the paperNot a substantial claim2 of 3 sources checked- cites [13] Eden, 2002, J. Clin. Investig. source
The Introduction reports that ARH1 defects caused impaired LDL receptor internalization in patients’ EBV-transformed lymphocytes, and the ‘LDL receptor function in ARH1-negative cells’ section reports defective internalization in monocyte-derived macrophages. The copy does not establish the claim for circulating, untransformed lymphocytes; it also notes some LDL uptake in macrophage cultures from one affected patient. No page number is provided for these passages. (LLM, conf=0.88)
Experimental — accuracy not yet measured - cites [25] Norman, 1999, J. Clin. Investig. source
The Analysis of LDL receptor function section reports that cells from two patients could bind LDL but could not internalize LDL receptors, supporting the endocytosis defect. The study used cultured lymphoblasts, not circulating lymphocytes or monocyte-derived macrophages, and our copy does not identify ARH as the required factor. No page number is provided in the indexed passages. (LLM, conf=0.88)
Experimental — accuracy not yet measured - cites [26] Wilund, 2002, Hum. Mol. Genet. source
- Findingcitation
In control untreated cells, the majority of ARH co-sedimented with vesicles containing EEA1 and Rab-4.
“As previously reported (15), in control untreated cells the majority of ARH co-sedimented with vesicles containing EEA1 and Rab-4 (Fig. 4A).”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [15] Jones, 2003, J. Biol. Chem. source
- Rests on an assumption: HepG2 cells are a valid model for human hepatocytes for studying ARH function in LDLR endocytosis.
- Findingown data
In non-confluent, non-polarized HepG2 cells, the ARH signal appears punctate and homogenously dispersed throughout the cytoplasm.
“As previously described for primary cultured fibroblasts and the HeLa epithelial cell line (11), we found that in non-confluent, non-polarized HepG2 cells the ARH signal appears punctate and homogenously dispersed throughout the cytoplasm (Fig. 1A).”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [11] Mishra, 2002, Proc. Natl. Acad. Sci. U. S. A. source
- Rests on an assumption: HepG2 cells are a valid model for human hepatocytes for studying ARH function in LDLR endocytosis.
- Findingown data§ Adaptor Protein ARH Is Recruited to the Plasma Membrane by Low Density Lipoprotein (LDL) Binding and Modulates Endocytosis of the LDL/LDL Receptor Complex in Hepatocytes *
Depletion of ARH caused about 80% reduction in LDL internalization.
“Quantitative immunofluorescence analysis revealed that the depletion of ARH caused about 80% reduction in LDL internalization.”
Quote in the paperSubstantial claim- Rests on an assumption: HepG2 cells are a valid model for human hepatocytes for studying ARH function in LDLR endocytosis.
- Findingown data
ARH protein partially colocalizes with LDLR-anti-LDLR complexes.
“Double immunofluorescence with anti-ARH polyclonal antibodies showed that ARH protein partially colocalizes with LDLRanti-LDLR complexes (Fig. 2C).”
Quote in the paperSubstantial claim- Rests on an assumption: HepG2 cells are a valid model for human hepatocytes for studying ARH function in LDLR endocytosis.
- Findingown data§ Adaptor Protein ARH Is Recruited to the Plasma Membrane by Low Density Lipoprotein (LDL) Binding and Modulates Endocytosis of the LDL/LDL Receptor Complex in Hepatocytes *
ARH is codistributed with LDLR on the basolateral area in confluent HepG2-polarized cells.
“Here we provide evidence that ARH is codistributed with LDLR on the basolateral area in confluent HepG2-polarized cells.”
- Also stated in: § The Intracellular Localization of ARH Protein Is Related to Cell Polarity-To analyze ARH expression · hedged
“In this experiment, ARH appeared to be colocalized with the receptor in the basolateral area of the cells (Fig. 1C).”
Quote in the paperSubstantial claim- Rests on an assumption: HepG2 cells are a valid model for human hepatocytes for studying ARH function in LDLR endocytosis.
- Also stated in: § The Intracellular Localization of ARH Protein Is Related to Cell Polarity-To analyze ARH expression · hedged
- Findingown data§ Adaptor Protein ARH Is Recruited to the Plasma Membrane by Low Density Lipoprotein (LDL) Binding and Modulates Endocytosis of the LDL/LDL Receptor Complex in Hepatocytes *
ARH is not constitutively associated with LDLR at the plasma membrane.
“Our results indicate that ARH is not constitutively associated with LDLR at the plasma membrane; instead, ARH is mainly recruited to the membrane after LDL binding, thus facilitating the endocytosis of the LDL-LDLR complex.”
Quote in the paperSubstantial claim- Rests on an assumption: HepG2 cells are a valid model for human hepatocytes for studying ARH function in LDLR endocytosis.
- Findingown data§ Adaptor Protein ARH Is Recruited to the Plasma Membrane by Low Density Lipoprotein (LDL) Binding and Modulates Endocytosis of the LDL/LDL Receptor Complex in Hepatocytes *
ARH is associated with other proteins of the endocytic machinery.
“Moreover, our findings indicate that ARH is associated with other proteins of the endocytic machinery.”
Quote in the paperNot a substantial claim- Rests on an assumption: HepG2 cells are a valid model for human hepatocytes for studying ARH function in LDLR endocytosis.
- Findingown data
ARH and DiI-LDL colocalized at the plasma membrane after treatment with DiI-LDL for 5 min at 37 °C.
“Double immunofluorescence with anti-ARH polyclonal antibodies showed that ARH and DiI-LDL colocalized at the plasma membrane (Fig. 2B), suggesting that ARH could be partially recruited to the plasma membrane after LDL binding to the LDLR.”
Quote in the paperSubstantial claim- Rests on an assumption: HepG2 cells are a valid model for human hepatocytes for studying ARH function in LDLR endocytosis.
- Findingcitation
ARH co-immunoprecipitates with β-adaptin and Dab2 in HepG2 cells.
“As already reported in the literature (14), we observed co-immunoprecipitation of the ARH protein with several components of the plasma membrane endocytic system, including the  subunit of the AP-2 complex (-adaptin) and the Dab2 protein.”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [14] He, 2002, J. Biol. Chem. source
- Rests on an assumption: Co-immunoprecipitation of ARH with endocytic proteins reflects their interaction in vivo.
- Findingown data
Only a small percentage of ARH was present in the same fractions as the LDLR in control untreated cells.
“Only a small percentage of ARH was present in the same fractions as the LDLR (Fig. 4A).”
Quote in the paperSubstantial claim- Rests on an assumption: HepG2 cells are a valid model for human hepatocytes for studying ARH function in LDLR endocytosis.
- Findingown data
ARH is associated with β-adaptin and Dab2.
“We found that ARH is associated with -adaptin and Dab2, in agreement with similar findings by Mishra et al. (11).”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [11] Mishra, 2002, Proc. Natl. Acad. Sci. U. S. A. source
- Rests on an assumption: Co-immunoprecipitation of ARH with endocytic proteins reflects their interaction in vivo.
- Findingown data
After 10 min, when LDL appeared exclusively in intracellular endocytic dots, no more ARH protein could be colocalized with the LDL-LDLR complex.
“After 10 min, when LDL appeared exclusively in intracellular endocytic dots, no more ARH protein could be colocalized with the LDL-LDLR complex.”
Quote in the paperSubstantial claim - Findingown data
The overlap of ARH protein with the LDL-LDLR complex at the level of the plasma membrane was almost negligible at 4 °C but became significant at 37 °C.
“In fact, we observed by immunofluorescence that the overlap of ARH protein with the LDL-LDLR complex at the level of the plasma membrane was almost negligible at 4 °C but became significant at 37 °C.”
Quote in the paperSubstantial claim - Findingown data
ARH co-immunoprecipitates with the small GTPase Rab-4 in HepG2 cells.
“In addition, we observed co-immunoprecipitation of ARH with the small GTPase Rab-4, which is a component of the early endosome and recycling arms of the endocytic pathway.”
Quote in the paperNot a substantial claim- Rests on an assumption: Co-immunoprecipitation of ARH with endocytic proteins reflects their interaction in vivo.
- Findingown data
Two minutes after LDL addition, ARH distribution showed a marked shift toward higher density fractions where LDLR was mainly localized.
“Conversely, 2 min after LDL addition, ARH distribution showed a marked shift toward higher density fractions where LDLR was mainly localized (Fig. 4B).”
Quote in the paperSubstantial claim- Rests on an assumption: HepG2 cells are a valid model for human hepatocytes for studying ARH function in LDLR endocytosis.
- Findingown data
In hepatocytes lacking ARH, most of the LDL resides on the cell surface.
“In fact, the immunofluorescence analysis revealed that in hepatocytes lacking ARH, most of the LDL resides on the cell surface.”
Quote in the paperSubstantial claim - Findingown data
No association was found in control untreated cells.
“As expected, no association was found in control untreated cells.”
Quote in the paperSubstantial claim- Rests on an assumption: HepG2 cells are a valid model for human hepatocytes for studying ARH function in LDLR endocytosis.
- Findingown data
Inhibition of LDL endocytosis was evident in 80% of HepG2 cells injected with ARH siRNA.
“Quantitative analysis performed by counting uninjected or injected cells presenting internalized LDL in five different areas of each slide randomly taken from three different experiments revealed that the inhibition of LDL endocytosis was evident in 80% of the cells injected with siRNA (Fig. 4D).”
Quote in the paperSubstantial claim - Findingown data
The loss of ARH arrested the LDLR trafficking cycle at the point of endocytosis from the cell surface in HepG2 cells.
“This is in contrast with our finding that the loss of ARH arrested the LDLR trafficking cycle at the point of endocytosis from the cell surface in HepG2 cells.”
Quote in the paperSubstantial claim - Findingown data
Rab-4 may colocalize with ARH.
“In addition, we observed that Rab-4 may colocalize with ARH.”
Quote in the paperNot a substantial claim- Rests on an assumption: Co-immunoprecipitation of ARH with endocytic proteins reflects their interaction in vivo.
- Findingown data
Clathrin-associated LDL-LDLR complexes showed co-immunoprecipitation with ARH transiently 2-5 min after the LDLR pathway was activated.
“These findings were corroborated by our subcellular fractionation and immunoprecipitation time course experiments, in which clathrin-associated LDL-LDLR complexes showed co-immunoprecipitation with ARH transiently 2-5 min after the LDLR pathway was activated.”
Quote in the paperSubstantial claim - Findingown data
ARH protein is recruited to the plasma membrane after stimulation of LDL internalization.
“The second major finding from our study is that ARH protein is recruited to plasma membrane after stimulation of LDL internalization.”
Quote in the paperSubstantial claim - Findingown data
The majority of ARH protein in hepatocytes appears in fractions where the LDLR receptor is absent.
“In particular, we observed that the majority of ARH protein in hepatocytes appears in fractions where the LDLR receptor is absent”
Quote in the paperSubstantial claim - Findingown data
No kinetics were detected in the associations between ARH and β-adaptin, Dab2, or Rab-4.
“We were unable to detect any kinetics in these associations.”
Quote in the paperNot a substantial claim- Rests on an assumption: Co-immunoprecipitation of ARH with endocytic proteins reflects their interaction in vivo.
- Findingown data
Western blot analysis using anti-ARH antibody revealed a positive band in samples treated with LDL for 2 min.
“Western blot analysis using the anti-ARH antibody revealed a positive band in samples treated with LDL for 2 min.”
Quote in the paperSubstantial claim- Rests on an assumption: HepG2 cells are a valid model for human hepatocytes for studying ARH function in LDLR endocytosis.
- Interpretationown data§ Adaptor Protein ARH Is Recruited to the Plasma Membrane by Low Density Lipoprotein (LDL) Binding and Modulates Endocytosis of the LDL/LDL Receptor Complex in Hepatocytes *
ARH protein is an important component of the endocytic machinery of LDLR in hepatocytes.
“Collectively, our data provide evidence that the ARH protein is an important component of the endocytic machinery of LDLR in hepatocytes.”
Quote in the paperSubstantial claim- Rests on an assumption: HepG2 cells are a valid model for human hepatocytes for studying ARH function in LDLR endocytosis.
- Interpretationcitation§ Adaptor Protein ARH Is Recruited to the Plasma Membrane by Low Density Lipoprotein (LDL) Binding and Modulates Endocytosis of the LDL/LDL Receptor Complex in Hepatocytes *
ARH is not constitutively associated with the LDLR-clathrin complex.
“clearly indicating that ARH is not constitutively associated with the LDLR-clathrin complex.”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [15] Jones, 2003, J. Biol. Chem. source
- Premisecitation§ Adaptor Protein ARH Is Recruited to the Plasma Membrane by Low Density Lipoprotein (LDL) Binding and Modulates Endocytosis of the LDL/LDL Receptor Complex in Hepatocytes *
This disorder, called Autosomal Recessive Hypercholesterolemia (ARH), is caused by mutations in the putative adaptor protein ARH.
“This disorder, called Autosomal Recessive Hypercholesterolemia (ARH), is caused by mutations in the putative adaptor protein ARH (9).”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [9] Garcia, 2001, Science. source
- Interpretationcitation§ Adaptor Protein ARH Is Recruited to the Plasma Membrane by Low Density Lipoprotein (LDL) Binding and Modulates Endocytosis of the LDL/LDL Receptor Complex in Hepatocytes *
ARH protein may be involved in the internalization of the LDL-LDLR complex.
“This strongly indicates that ARH protein may be involved in the internalization of the LDL-LDLR complex (13).”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [13] Eden, 2002, J. Clin. Investig. source
- Premisecitation§ Adaptor Protein ARH Is Recruited to the Plasma Membrane by Low Density Lipoprotein (LDL) Binding and Modulates Endocytosis of the LDL/LDL Receptor Complex in Hepatocytes *
Elucidation of the specific role of ARH in the LDLR endocytosis has been hampered by the fact that cultured skin fibroblasts from ARH patients do not show major defects in LDL uptake and degradation.
“Elucidation of the specific role of ARH in the LDLR endocytosis has been hampered by the fact that cultured skin fibroblasts from ARH patients do not show major defects in LDL uptake and degradation (12).”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [12] Arca, 2002, Lancet. source
- Study design and conductown data§ Adaptor Protein ARH Is Recruited to the Plasma Membrane by Low Density Lipoprotein (LDL) Binding and Modulates Endocytosis of the LDL/LDL Receptor Complex in Hepatocytes *
Twenty-four hours after transfection with ARH-specific RNA interference, ARH protein was depleted in HepG2 cells by more than 70%.
“Twenty-four hours after transfection with ARH-specific RNA interference, ARH protein was depleted in HepG2 cells by more than 70%.”
Quote in the paperSubstantial claim- Rests on an assumption: HepG2 cells are a valid model for human hepatocytes for studying ARH function in LDLR endocytosis.
- Premisecitation§ Adaptor Protein ARH Is Recruited to the Plasma Membrane by Low Density Lipoprotein (LDL) Binding and Modulates Endocytosis of the LDL/LDL Receptor Complex in Hepatocytes *
Immunoprecipitation experiments demonstrated that, at the steady state, the majority of ARH protein appears in fractions from which the LDLR receptor is absent.
“Moreover, immunoprecipitation experiments demonstrated that, at the steady state, the majority of ARH protein appears in fractions from which the LDLR receptor is absent (15), clearly indicating that ARH is not constitutively associated with the LDLR-clathrin complex.”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [15] Jones, 2003, J. Biol. Chem. source
- Interpretationcitation§ Adaptor Protein ARH Is Recruited to the Plasma Membrane by Low Density Lipoprotein (LDL) Binding and Modulates Endocytosis of the LDL/LDL Receptor Complex in Hepatocytes *
It is unlikely that an inappropriate anchorage of LDLR in coated pits is the major cause of LDLR malfunction in arh-/- hepatocytes.
“Therefore, it is unlikely that an inappropriate anchorage of LDLR in coated pits is the major cause of LDLR malfunction in arhϪ/Ϫ hepatocytes.”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [17] Pathak, 1990, J. Cell Biol. source
- Premisecitation§ Adaptor Protein ARH Is Recruited to the Plasma Membrane by Low Density Lipoprotein (LDL) Binding and Modulates Endocytosis of the LDL/LDL Receptor Complex in Hepatocytes *
Experiments performed in ARH-deficient mice demonstrated that ARH does not direct LDLR to the sinusoidal membrane.
“Experiments performed in ARH-deficient mice demonstrated that this was not the case.”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [15] Jones, 2003, J. Biol. Chem. source
- Premisecitation§ Adaptor Protein ARH Is Recruited to the Plasma Membrane by Low Density Lipoprotein (LDL) Binding and Modulates Endocytosis of the LDL/LDL Receptor Complex in Hepatocytes *
Individuals lacking ARH have severe hypercholesterolemia due to an impaired hepatic clearance of LDL.
“Individuals lacking ARH have severe hypercholesterolemia due to an impaired hepatic clearance of LDL.”
Quote in the paperSubstantial claim0 of 2 sources checked - Premisecitation§ Adaptor Protein ARH Is Recruited to the Plasma Membrane by Low Density Lipoprotein (LDL) Binding and Modulates Endocytosis of the LDL/LDL Receptor Complex in Hepatocytes *
In arh-/- mouse livers, LDLR appears to be normally sorted to the sinusoidal surface.
“In arh Ϫ/Ϫ mouse livers, LDLR appears to be normally sorted to the sinusoidal surface (15).”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [15] Jones, 2003, J. Biol. Chem. source
- Premisecitation§ Adaptor Protein ARH Is Recruited to the Plasma Membrane by Low Density Lipoprotein (LDL) Binding and Modulates Endocytosis of the LDL/LDL Receptor Complex in Hepatocytes *
ARH is a newly discovered adaptor protein required for the efficient activity of LDLR in selected tissues.
“ARH is a newly discovered adaptor protein required for the efficient activity of low density lipoprotein receptor (LDLR) in selected tissues.”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [9] Garcia, 2001, Science. source
- Interpretationown data
ARH protein is required to promote the internalization of the LDL-LDLR complex into hepatocytes.
“In summary, our findings demonstrate that ARH protein is required to promote the internalization of the LDL-LDLR complex into hepatocytes.”
Quote in the paperSubstantial claim- Rests on an assumption: The siRNA-mediated loss of ARH in HepG2 cells mimics the in vivo pathological loss of function of hepatic LDL uptake in ARH patients.
- Interpretationreasoning
ARH may associate with endocytic accessory proteins of the early endosome compartment.
“These observations strongly suggest that ARH may associate with endocytic accessory proteins of the early endosome compartment.”
Quote in the paperSubstantial claim- Rests on an assumption: Co-immunoprecipitation of ARH with endocytic proteins reflects their interaction in vivo.
- Interpretationown data
ARH could be partially recruited to the plasma membrane after LDL binding to the LDLR.
“Double immunofluorescence with anti-ARH polyclonal antibodies showed that ARH and DiI-LDL colocalized at the plasma membrane (Fig. 2B), suggesting that ARH could be partially recruited to the plasma membrane after LDL binding to the LDLR.”
Quote in the paperSubstantial claim- Rests on an assumption: HepG2 cells are a valid model for human hepatocytes for studying ARH function in LDLR endocytosis.
- Interpretationreasoning
At steady state, ARH appears to be colocalized with endocytic accessory proteins in liver cells.
“At steady state, ARH appears to be colocalized with endocytic accessory proteins in the liver cells.”
Quote in the paperNot a substantial claim- Rests on an assumption: Co-immunoprecipitation of ARH with endocytic proteins reflects their interaction in vivo.
- Interpretationreasoning
In hepatocytes of ARH patients, defective function of this sorting adaptor may lead to faulty LDLR traffic and hypercholesterolemia.
“In hepatocytes of ARH patients, defective function of this sorting adaptor may lead to faulty LDLR traffic and hypercholesterolemia.”
Quote in the paperSubstantial claim - Interpretationown data
ARH could be involved in the early steps of LDLR endocytosis.
“These results indicate that ARH could be involved in the early steps of LDLR endocytosis.”
Quote in the paperSubstantial claim- Rests on an assumption: HepG2 cells are a valid model for human hepatocytes for studying ARH function in LDLR endocytosis.
- Interpretationown data
The adaptor protein ARH plays a crucial role in the internalization of LDLR by hepatocytes.
“The results of this study demonstrate that the adaptor protein ARH plays a crucial role in the internalization of LDLR by hepatocytes.”
Quote in the paperSubstantial claim- Rests on an assumption: The siRNA-mediated loss of ARH in HepG2 cells mimics the in vivo pathological loss of function of hepatic LDL uptake in ARH patients.
- Interpretationown data
ARH protein plays an essential role in the LDLR endocytic process in HepG2 cells.
“This suggests that, at least in HepG2 cells, ARH protein plays an essential role in the LDLR endocytic process.”
Quote in the paperSubstantial claim - Interpretationown data
ARH is recruited to LDLR-containing early endocytic compartments.
“Those results indicated that ARH is recruited to LDLR-containing early endocytic compartments.”
Quote in the paperSubstantial claim - Interpretationown data
Activation of LDL internalization is the signal that induces movement and recruitment of ARH.
“Our immunofluorescent experiments carried out using anti-LDLR C7, a non-physiological ligand containing neither apolipoprotein B nor lipids, clearly suggest that the activation of LDL internalization is the signal that induces movement and recruitment of ARH.”
Quote in the paperSubstantial claim- Rests on an assumption: Anti-LDLR C7, a non-physiological ligand lacking apolipoprotein B and lipids, activates LDL internalization in a way that validly indicates the signal for ARH recruitment.
- Interpretationown data
These findings are consistent with ARH being recruited to LDLR-containing early endocytic vesicles when LDLR internalization is stimulated.
“These findings are consistent with ARH being recruited to LDLR-containing early endocytic vesicles when LDLR internalization is stimulated.”
Quote in the paperSubstantial claim- Rests on an assumption: HepG2 cells are a valid model for human hepatocytes for studying ARH function in LDLR endocytosis.
- Interpretationown data
Silencing of ARH was sufficient to functionally block DiI-LDL internalization in HepG2 cells.
“Thus, the silencing of ARH was sufficient to functionally block DiI-LDL internalization.”
Quote in the paperSubstantial claim
- It may state two separate claims as one. 27
- Probably not a substantial claim. 20
It may state two separate claims as one.
Findingown dataIn confluent HepG2 monolayers, the ARH signal is punctate but prevalently distributed in the basolateral regions, and to a lesser extent evident in the proximity of the apical poles.
“The ARH signal in these cells is also punctate but prevalently distributed in the basolateral regions, and to a lesser extent evident in the proximity of the apical poles (Fig. 1B, arrow).”
Quote in the paperNot a substantial claim- Rests on an assumption: HepG2 cells are a valid model for human hepatocytes for studying ARH function in LDLR endocytosis.
It may state two separate claims as one.
Findingown data§ Adaptor Protein ARH Is Recruited to the Plasma Membrane by Low Density Lipoprotein (LDL) Binding and Modulates Endocytosis of the LDL/LDL Receptor Complex in Hepatocytes *Up-regulation of LDLR expression by serum starvation does not modify ARH and/or LDLR distribution or induce colocalization of ARH with LDLRs on the plasma membrane.
“This distribution is not modified by the overexpression of LDLR.”
- Also stated in: § The Intracellular Localization of ARH Protein Is Related to Cell Polarity-To analyze ARH expression · plain
“Although these conditions increase the presence of receptors on the cell surface, they did not modify the ARH and/or LDLR distribution or induce a colocalization of ARH with LDLRs on the plasma membrane (Fig. 2A).”
Quote in the paperSubstantial claim- Rests on an assumption: HepG2 cells are a valid model for human hepatocytes for studying ARH function in LDLR endocytosis.
- Also stated in: § The Intracellular Localization of ARH Protein Is Related to Cell Polarity-To analyze ARH expression · plain
It may state two separate claims as one.
Findingown dataColocalization of ARH with fluorescent LDL at the plasma membrane appeared maximal after 2 min at 37 °C and rapidly decreased, although it was still evident after 5 min.
“In fact, colocalization of ARH with fluorescent LDL at the level of the plasma membrane appeared maximal after 2 min at 37 °C (Fig. 3) and rapidly decreased, although it was still evident after 5 min (Fig. 3) when LDL appeared also in intracellular endocytic dots.”
Quote in the paperSubstantial claim- Rests on an assumption: HepG2 cells are a valid model for human hepatocytes for studying ARH function in LDLR endocytosis.
It may state two separate claims as one.
Findingown dataTransient loss of ARH by siRNA resulted in failure of LDL endocytosis and almost complete absence of the LDL-LDLR complex in the internal compartment of hepatocytes.
“Transient loss of ARH by siRNA resulted in the failure of LDL endocytosis and the almost complete absence of the LDL-LDLR complex in the internal compartment of hepatocytes.”
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.
Findingown dataAt 48 h after siRNA transfection, ARH protein levels showed a slight increase, and this increase was more evident at 72 h.
“The lysates obtained 48 h after incubation with siRNA showed a slight increase of ARH, and this was more evident 72 h after transfection.”
Quote in the paperNot a substantial claimIt may state two separate claims as one.
Findingown dataIn HepG2 cells microinjected with ARH siRNA and dextran-FITC, DiI-LDL internalization was blocked, with no intracellular LDL-positive dots visible, whereas uninjected cells or cells injected with dextran-FITC alone showed DiI-LDL concentrated in intracellular endocytic dots.
“In uninjected cells and cells injected only with dextran-FITC as a control, DiI-LDL was concentrated in intracellular endocytic dots, whereas in cells microinjected with siRNA and dextran-FITC mixture no intracellular LDL-positive dots were visible (Fig. 4C).”
Quote in the paperSubstantial claimIt may state two separate claims as one.
Findingown dataTransfection of HepG2 cells with an siRNA duplex targeting segment 88-112 of the ARH open reading frame reduced ARH protein levels by approximately 70-80% at 24 h, while actin levels changed only slightly.
“Twenty-four hours after the transfection, the amounts of ARH protein were strongly reduced (ϳ70 -80% decrease), whereas the amount of actin had changed only slightly.”
Quote in the paperSubstantial claimIt may state two separate claims as one.
Findingown dataThe band was still evident after 5 min of treatment and declined thereafter.
“The band was still evident after 5 min of treatment and declined thereafter.”
Quote in the paperSubstantial claim- Rests on an assumption: HepG2 cells are a valid model for human hepatocytes for studying ARH function in LDLR endocytosis.
It may state two separate claims as one.
Findingown dataThe pattern of co-immunoprecipitation of ARH with β-adaptin, Dab2, and Rab-4 was already present at 4 °C and was not apparently modified at later times after LDL addition.
“It is interesting to note that the pattern of co-immunoprecipitation of ARH with -adaptin, Dab2, and Rab-4 was already present at 4 °C and was not apparently modified at later times after LDL addition.”
Quote in the paperNot a substantial claim- Rests on an assumption: Co-immunoprecipitation of ARH with endocytic proteins reflects their interaction in vivo.
It may state two separate claims as one.
Interpretationown data§ Adaptor Protein ARH Is Recruited to the Plasma Membrane by Low Density Lipoprotein (LDL) Binding and Modulates Endocytosis of the LDL/LDL Receptor Complex in Hepatocytes *ARH is an endocytic sorting adaptor that actively participates in the internalization of the LDL-LDLR complex, possibly enhancing the efficiency of its packaging into the endocytic vesicles.
“We suggest that ARH is an endocytic sorting adaptor that actively participates in the internalization of the LDL-LDLR complex, possibly enhancing the efficiency of its packaging into the endocytic vesicles.”
Quote in the paperSubstantial claim- Rests on an assumption: HepG2 cells are a valid model for human hepatocytes for studying ARH function in LDLR endocytosis.
It may state two separate claims as one.
Premisecitation§ Adaptor Protein ARH Is Recruited to the Plasma Membrane by Low Density Lipoprotein (LDL) Binding and Modulates Endocytosis of the LDL/LDL Receptor Complex in Hepatocytes *LDLRs are dispersed on the plasma membrane of hepatocytes but clustered in coated pits on fibroblasts.
“It must be noted, however, that LDLRs are dispersed on the plasma membrane of hepatocytes but clustered in coated pits on fibroblasts (17).”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [17] Pathak, 1990, J. Cell Biol. source
Probably not a substantial claim. The paper’s conclusions may not depend on it.
Premisecitation§ Adaptor Protein ARH Is Recruited to the Plasma Membrane by Low Density Lipoprotein (LDL) Binding and Modulates Endocytosis of the LDL/LDL Receptor Complex in Hepatocytes *LDLR plays a pivotal role in the regulation of cholesterol metabolism.
“The low density lipoprotein receptor (LDLR) 2 plays a pivotal role in the regulation of cholesterol metabolism (1).”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [1] Brown, 1986, Science. source
It may state two separate claims as one.
Premisecitation§ Adaptor Protein ARH Is Recruited to the Plasma Membrane by Low Density Lipoprotein (LDL) Binding and Modulates Endocytosis of the LDL/LDL Receptor Complex in Hepatocytes *The majority of studies so far available have been carried out on lymphocytes from ARH patients, in which the LDLR function is significantly impaired.
“Therefore, the majority of studies so far available have been carried out on lymphocytes from ARH patients, in which the LDLR function is significantly impaired (13,14).”
Quote in the paperSubstantial claim0 of 2 sources checkedProbably not a substantial claim. The paper’s conclusions may not depend on it.
Premisecitation§ Adaptor Protein ARH Is Recruited to the Plasma Membrane by Low Density Lipoprotein (LDL) Binding and Modulates Endocytosis of the LDL/LDL Receptor Complex in Hepatocytes *Early studies on normal and FH fibroblasts demonstrated that LDLRs, shortly after being synthesized, appear on the cell surface where they gather in coated pits.
“Early studies on normal and FH fibroblasts demonstrated that LDLRs, shortly after being synthesized, appear on the cell surface where they gather in coated pits (1).”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [1] Brown, 1986, Science. source
It may state two separate claims as one.
Premisecitation§ Adaptor Protein ARH Is Recruited to the Plasma Membrane by Low Density Lipoprotein (LDL) Binding and Modulates Endocytosis of the LDL/LDL Receptor Complex in Hepatocytes *Although LDL degradation appears to be markedly reduced in lymphocytes from ARH patients, cell surface LDL binding is increased.
“Moreover, although LDL degradation appears to be markedly reduced in lymphocytes, cell surface LDL binding is increased.”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [13] Eden, 2002, J. Clin. Investig. source
It may state two separate claims as one.
Interpretationown data§ Adaptor Protein ARH Is Recruited to the Plasma Membrane by Low Density Lipoprotein (LDL) Binding and Modulates Endocytosis of the LDL/LDL Receptor Complex in Hepatocytes *ARH is mainly recruited to the membrane after LDL binding, thus facilitating the endocytosis of the LDL-LDLR complex.
“Our results indicate that ARH is not constitutively associated with LDLR at the plasma membrane; instead, ARH is mainly recruited to the membrane after LDL binding, thus facilitating the endocytosis of the LDL-LDLR complex.”
Quote in the paperSubstantial claim- Rests on an assumption: HepG2 cells are a valid model for human hepatocytes for studying ARH function in LDLR endocytosis.
Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.
Premisecitation§ Adaptor Protein ARH Is Recruited to the Plasma Membrane by Low Density Lipoprotein (LDL) Binding and Modulates Endocytosis of the LDL/LDL Receptor Complex in Hepatocytes *The phosphotyrosine-binding sequence also binds inositol phospholipids, which may anchor the protein to the plasma membrane.
“The phosphotyrosine-binding sequence also binds inositol phospholipids, which may anchor the protein to the plasma membrane (11).”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [11] Mishra, 2002, Proc. Natl. Acad. Sci. U. S. A. source
Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.
Premisecitation§ Adaptor Protein ARH Is Recruited to the Plasma Membrane by Low Density Lipoprotein (LDL) Binding and Modulates Endocytosis of the LDL/LDL Receptor Complex in Hepatocytes *ARH is required for the efficient internalization of the LDL-LDLR complex and to stabilize the association of the receptor with LDL in Epstein-Barr virus-immortalized B lymphocytes.
“It has been demonstrated that ARH is required for the efficient internalization of the LDL-LDLR complex and to stabilize the association of the receptor with LDL in Epstein-Barr virus-immortalized B lymphocytes.”
Quote in the paperNot a substantial claim0 of 2 sources checkedProbably not a substantial claim. The paper’s conclusions may not depend on it.
Premisecitation§ Adaptor Protein ARH Is Recruited to the Plasma Membrane by Low Density Lipoprotein (LDL) Binding and Modulates Endocytosis of the LDL/LDL Receptor Complex in Hepatocytes *Coated pits are specialized regions of cell membranes that are lined on the cytoplasmic surface by a protein called clathrin.
“These are specialized regions of cell membranes that are lined on the cytoplasmic surface by a protein called clathrin (3).”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [3] Goldstein, 1979, Nature. source
Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.
Premisecitation§ Adaptor Protein ARH Is Recruited to the Plasma Membrane by Low Density Lipoprotein (LDL) Binding and Modulates Endocytosis of the LDL/LDL Receptor Complex in Hepatocytes *FH is inherited as a co-dominant trait, and affected subjects show a markedly impaired LDLR function because of mutations in the LDLR gene.
“FH is inherited as a co-dominant trait (2), and affected subjects show a markedly impaired LDLR function because of mutations in the LDLR gene (2).”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [2] Goldstein, 2001
Couldn’t be matched to a paper in Crossref, so it can’t be checked.
Probably not a substantial claim. The paper’s conclusions may not depend on it.
Premisecitation§ Adaptor Protein ARH Is Recruited to the Plasma Membrane by Low Density Lipoprotein (LDL) Binding and Modulates Endocytosis of the LDL/LDL Receptor Complex in Hepatocytes *siRNA methodology has been demonstrated to be the most powerful way to selectively reduce the intracellular concentration of proteins.
“This has been demonstrated to be the most powerful way to selectively reduce the intracellular concentration of proteins (18,19).”
Quote in the paperNot a substantial claim0 of 2 sources checkedProbably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.
Premisecitation§ Adaptor Protein ARH Is Recruited to the Plasma Membrane by Low Density Lipoprotein (LDL) Binding and Modulates Endocytosis of the LDL/LDL Receptor Complex in Hepatocytes *Very quickly, the clathrin coat dissociates and multiple endocytic vesicles fuse to create endosomes.
“Very quickly, the clathrin coat dissociates and multiple endocytic vesicles fuse to create endosomes (4 -6).”
Quote in the paperNot a substantial claim0 of 3 sources checkedProbably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.
Premisecitation§ Adaptor Protein ARH Is Recruited to the Plasma Membrane by Low Density Lipoprotein (LDL) Binding and Modulates Endocytosis of the LDL/LDL Receptor Complex in Hepatocytes *At the acid pH in endosomes, the LDL dissociates from the receptor, which returns to the surface to initiate another cycle of endocytosis.
“At the acid pH in endosomes, the LDL dissociates from the receptor, which returns to the surface to initiate another cycle of endocytosis (7,8).”
Quote in the paperNot a substantial claim0 of 2 sources checkedIt may state two separate claims as one.
Interpretationown data§ Adaptor Protein ARH Is Recruited to the Plasma Membrane by Low Density Lipoprotein (LDL) Binding and Modulates Endocytosis of the LDL/LDL Receptor Complex in Hepatocytes *Activation of LDLR-mediated endocytosis, but not the binding of LDL to LDLR, promotes a significant colocalization of ARH with the LDL-LDLR complex that peaked at 2 min at 37 °C.
“Conversely, the activation of the LDLR-mediated endocytosis, but not the binding of LDL to LDLR, promotes a significant colocalization of ARH with LDL-LDLR complex that peaked at 2 min at 37 °C.”
Quote in the paperSubstantial claim- Rests on an assumption: HepG2 cells are a valid model for human hepatocytes for studying ARH function in LDLR endocytosis.
It may state two separate claims as one.
Premisecitation§ Adaptor Protein ARH Is Recruited to the Plasma Membrane by Low Density Lipoprotein (LDL) Binding and Modulates Endocytosis of the LDL/LDL Receptor Complex in Hepatocytes *It has been observed that the distribution of the immunodetectable LDLR is significantly altered in lymphocytes from ARH patients and that it mostly resides on the plasma membrane.
“It has been observed that the distribution of the immunodetectable LDLR is significantly altered in these cells and that it mostly resides on the plasma membrane.”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [13] Eden, 2002, J. Clin. Investig. source
Probably not a substantial claim.
Study design and conductown dataARH protein reduction was quantitated by immunoblotting with anti-ARH antibodies and anti-actin antibodies as a loading control.
“To quantitate the reduction of ARH expression, the cells were lysed 24, 48, and 72 h after transfection and analyzed by immunoblotting with anti-ARH or anti-actin antibodies as a control (Fig. 6B).”
Quote in the paperSubstantial claimProbably not a substantial claim.
PremisecitationAnti-LDLR C7 is known to induce internalization and recycling of LDLR similarly to LDL particles.
“Anti-LDLR C7 is known to induce internalization and recycling of LDLR similarly to LDL particles (24).”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [24] Beisiegel, 1981, J. Biol. Chem. source
Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.
PremisecitationUptakes of [3H]cholesteryl oleyl ether-labeled LDL, 125I-LDL, and Dil-LDL were all normal when arh−/− hepatocytes were cultured.
“Surprisingly, the same authors reported that uptakes of [ 3 H]cholesteryl oleyl ether-labeled LDL, 125 I-LDL, and Dil-LDL were all normal when arhϪ/Ϫ hepatocytes were cultured.”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [28] Harada-Shiba, 2004, Circ. Res. source
It may state two separate claims as one.
InterpretationreasoningARH acts as a molecular usher, gathering the LDL-LDLR complex into clathrin-coated vesicles and presumably enhancing the efficiency of vesicle packaging.
“Therefore, ARH appears to act as a molecular usher, gathering the LDL-LDLR complex into the clathrin-coated vesicles and presumably enhancing the efficiency of vesicle packaging.”
Quote in the paperSubstantial claimProbably not a substantial claim. The paper’s conclusions may not depend on it.
Study design and conductown dataThe immunoprecipitation experiments included a negative control without cell lysate.
“Lane 5 is a negative control experiment where 25 l of Protein G/Protein A-Sepharose beads were incubated with anti--adaptin at 4 °C overnight without cell lysate.”
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.
Study design and conductown dataThe siRNA knockdown experiments in HepG2 cells included a scrambled-sequence positive control and a transfection-reagent negative control.
“Cells were transfected with siRNA, or with duplex of unrelated scrambled sequence as a positive control, or with transfection reagent alone as negative control.”
Quote in the paperNot a substantial claimProbably not a substantial claim.
PremisecitationLDLR tends to cluster in clathrin-coated pits on the cell surface under defined conditions.
“As noted in the Introduction, LDLR tends to cluster in clathrincoated pits on the cell surface under defined conditions.”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [1] Brown, 1986, Science. source
It may state two separate claims as one.
InterpretationreasoningAfter the LDL-LDLR complex has been formed, its internalization is promoted by the juxtaposition of ARH to the complex, and by this mechanism LDL fluxes through early endosomes.
“After LDL-LDLR complex has been formed, its internalization is promoted by the juxtaposition of ARH to the complex, and by this mechanism LDL fluxes through early endosomes.”
Quote in the paperSubstantial claimIt may state two separate claims as one.
PremisecitationLymphocytes obtained from ARH-negative patients did not accumulate LDL-gold complex in multivesicular bodies characteristic of late endosomes and lysosomes, confirming lack of internalization of LDL in these cells.
“More recently, it has been observed that lymphocytes obtained from ARH-negative patients did not accumulate LDL-gold complex in multivesicular bodies characteristic of late endosomes and lysosomes, thus confirming the lack of internalization of LDL in these cells (27).”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [27] Michaely, 2004, J. Biol. Chem. source
Probably not a substantial claim.
Study design and conductown dataQuantitative analysis of DiI-LDL internalization counted cells in five randomly taken areas per slide from three different experiments.
“Quantitative analysis performed by counting uninjected or injected cells presenting internalized LDL in five different areas of each slide randomly taken from three different experiments”
Quote in the paperSubstantial claimProbably not a substantial claim. The paper’s conclusions may not depend on it.
Study design and conductnone givenSelected siRNA sequences were submitted to BLAST searches against the human genome sequence to ensure that only the desired mRNA was targeted.
“Selected sequences were submitted to BLAST searches against the human genome sequence to ensure that only the desired mRNA was targeted.”
Quote in the paperNot a substantial claimProbably not a substantial claim. The paper’s conclusions may not depend on it.
Study design and conductnone givenTo ensure that bile canaliculus-like regions were formed de novo during the culture period, cells were vigorously separated before plating.
“To ensure that bile canaliculus-like regions were formed de novo during the culture period, cells were vigorously separated before plating.”
Quote in the paperNot a substantial claimProbably not a substantial claim. The paper’s conclusions may not depend on it.
PremisecitationAfter in vivo injection of [3H]cholesteryl oleyl ether-labeled LDL, less radioactivity is recovered from the liver of ARH-deficient mice than from livers of wild-type mice.
“In a recent study, Harada-Shiba et al. (28) observed that after in vivo injection of [ 3 H]cholesteryl oleyl ether-labeled LDL, less radioactivity is recovered from the liver of ARH-deficient mice than from livers of wild-type mice.”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [28] Harada-Shiba, 2004, Circ. Res. source
It may state two separate claims as one.
PremisecitationHepatic clearance of LDL from plasma is markedly retarded in arh−/− mice as well as in ARH patients.
“On the other hand, our observations concur with the evidence that hepatic clearance of LDL from plasma is markedly retarded in arhϪ/Ϫ mice as well as in ARH patients (12,15).”
Quote in the paperNot a substantial claim0 of 2 sources checked
The paper relies on this without support.
Little information is available on the role of ARH in liver cells.
“However, little information is available on the role of ARH in liver cells.”
“How ARH works in hepatocytes is less known.”
The paper relies on this without support.
HepG2 cells are a valid model for human hepatocytes for studying ARH function in LDLR endocytosis.
“To analyze ARH expression and distribution in human hepatocytes, we investigated the intracellular localization of ARH protein and the possible colocalization with LDLR in HepG2 cells, a hepatoma cell line that grows to subconfluent monolayers in 12 h or to confluent polarized monolayers in 72 h.”
“We further investigated this possibility in the human hepatocyte cell line HepG2.”
The paper relies on this without support.
The siRNA-mediated loss of ARH in HepG2 cells mimics the in vivo pathological loss of function of hepatic LDL uptake in ARH patients.
“This strategy could mimic the in vivo pathological loss of function of the hepatic LDL uptake in ARH patients.”
The paper relies on this without support.
Anti-LDLR C7, a non-physiological ligand lacking apolipoprotein B and lipids, activates LDL internalization in a way that validly indicates the signal for ARH recruitment.
“Our immunofluorescent experiments carried out using anti-LDLR C7, a non-physiological ligand containing neither apolipoprotein B nor lipids, clearly suggest that the activation of LDL internalization is the signal that induces movement and recruitment of ARH.”
The paper relies on this without support.
Co-immunoprecipitation of ARH with endocytic proteins reflects their interaction in vivo.
“To demonstrate interaction of ARH with other proteins involved in the clathrin-mediated internalization process in vivo, we carried out immunoprecipitations with anti--adaptin, anti-Dab2, and anti-Rab-4 followed by immunoblotting for ARH.”
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.
LDL internalization results cite the wrong figure
MinorThe text directs readers to Fig. 4C for the microinjection result, but Figure 4 is the sucrose-gradient analysis. The microinjection result is labeled Figure 6C; the same passage also cites Fig. 4D for a quantitative result labeled Figure 6D.
“whereas in cells microinjected with siRNA and dextran-FITC mixture no intracellular LDL-positive dots were visible (Fig. 4C).”
“Figure: 6 FIGURE 6 . FIGURE6.Effect of the microinjection of siRNA for ARH on LDL internalization.A, sequence of the siRNA used for the microinjection experiments.”
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Metadata
- Authors
- Sirinian, M. I., Belleudi, F., Campagna, F., Ceridono, M., Garofalo, T., Quagliarini, F., Verna, R., Calandra, S., Bertolini, S., Sorice, M., Torrisi, M. R., & Arca, M.
- Affiliations
- Sapienza University of Rome (IT)
- Azienda Ospedaliera Sant'Andrea (IT)
- University of Modena and Reggio Emilia (IT)
- University of Genoa (IT)
- Corresponding author
- Arca, Marcello
- ORCID
- Venue
- Journal of Biological Chemistry
- Publisher
- Elsevier BV
- Published
- 2005-11
- Volume
- 280
- Issue
- 46
- Pages
- 38416-38423
- ISSN
- 0021-9258 (ISSN-L)
- OpenAlex
- W2171519536
- Semantic Scholar
- a0faeff2f071d1382739245f01b0a1d7b2c107a1
- Cited by
- 42
- References
- 33
- Citations by year
- 2025: 2 · 2023: 1 · 2022: 3 · 2021: 3 · 2020: 2 · 2019: 2 · 2017: 3 · 2016: 2 · 2015: 4 · 2014: 3 · 2013: 1 · 2012: 1
- FWCI
- 0.87
- Type
- journal article
- Language
- en
- Open access
- hybrid
- doi.org
- Licence
- Locations
- Journal of Biological Chemistry (publishedVersion, cc-by)
- PubMed (publishedVersion)
- IRIS UNIMORE (University of Modena and Reggio Emilia) (submittedVersion, cc-by)
- CINECA IRIS Institutial Research Information System (University of Genoa) (submittedVersion)
- IRIS Research product catalog (Sapienza University of Rome) (submittedVersion)
- Funders
- Ministero della Salute
- Associazione Italiana per la Ricerca sul Cancro
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Biochemistry › Cholesterol and Lipid Metabolism
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Cellular transport and secretion
- Health Sciences › Medicine › Cardiology and Cardiovascular Medicine › Lipoproteins and Cardiovascular Health
- Keywords
- LDL receptor, LDL internalization, receptor-mediated endocytosis, plasma membrane recruitment, hypercholesterolemia, endocytic sorting, endocytic machinery, HepG2 cells, RNAi knockdown
- MeSH terms
- B-Lymphocytes, Base Sequence, Biochemistry, Cell Line, Cell Membrane, Centrifugation, Density Gradient, Clathrin, Endocytosis, Herpesvirus 4, Human, Humans, Lipoproteins, LDL, Liver, Microscopy, Fluorescence, Molecular Sequence Data, Protein Binding, Receptors, LDL, Sucrose, Temperature, Time Factors, Blotting, Western, Microscopy, Confocal, Gene Silencing, Hepatocytes, RNA Interference, RNA, Small Interfering, Immunoprecipitation, Adaptor Proteins, Signal Transducing
About these checks
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).
Cited sources are checked with a language model’s help (gpt-6-sol).
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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