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WIPI-dependent autophagy during neutrophil differentiation of NB4 acute promyelocytic leukemia cells

Brigger, D, Proikas-Cezanne, T, Tschan, M P·2014·Cell Death & Disease
DOI 10.1038/cddis.2014.261PMC PMC4123064
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Figure 1 Significantly decreased WIPI-1 mRNA expression in primary AML patient samples and increase in WIPI-1 expression upon ATRA treatment of NB4 APL cells was detected. (a) WIPI-1 mRNA levels of granulocytes from healthy donors and AML patient samples were measured using qPCR. Data represent log2 expression levels and were normalized to the expression levels of the two housekeeping genes HMBS and ABL. For better readability, we multiplied the results by (−1) and excluded Ct values higher than 40 (ΔCt=40−CtWIPI-1−(Mean CtHMBS and CtABL1) × (−1)). (b) mRNA levels of WIPI-1 expression was measured in control, and ATRA-treated NB4, NB4-R2 APL cell lines at day 4 using qPCR. Values were normalized to the housekeeping gene HMBS and given as n-fold mRNA expression relative to the control of day 4. (c, d) Increase in endogenous WIPI-1 puncta was detected using confocal microscopy and was further quantified using ImageJ software. (e) PI3P:WIPI-1 binding assay of cell lysates from NB4 cells treated with 1 μM ATRA for 6 days. Each membrane was loaded with equal total protein lysate. M.W.U, *P<0.05. **P<0.01, ***P<0.001. M.W.U, Mann–Whitney U-test
Figure 1 Significantly decreased WIPI-1 mRNA expression in primary AML patient samples and increase in WIPI-1 expression upon ATRA treatment of NB4 APL cells was detected. (a) WIPI-1 mRNA levels of granulocytes from healthy donors and AML patient samples were measured using qPCR. Data represent log2 expression levels and were normalized to the expression levels of the two housekeeping genes HMBS and ABL. For better readability, we multiplied the results by (−1) and excluded Ct values higher than 40 (ΔCt=40−CtWIPI-1−(Mean CtHMBS and CtABL1) × (−1)). (b) mRNA levels of WIPI-1 expression was measured in control, and ATRA-treated NB4, NB4-R2 APL cell lines at day 4 using qPCR. Values were normalized to the housekeeping gene HMBS and given as n-fold mRNA expression relative to the control of day 4. (c, d) Increase in endogenous WIPI-1 puncta was detected using confocal microscopy and was further quantified using ImageJ software. (e) PI3P:WIPI-1 binding assay of cell lysates from NB4 cells treated with 1 μM ATRA for 6 days. Each membrane was loaded with equal total protein lysate. M.W.U, *P<0.05. **P<0.01, ***P<0.001. M.W.U, Mann–Whitney U-testCC BY-NC-SA · PubMed Central
Figure 2 PU.1-dependent WIPI-1 regulation in NB4 APL cells. (a) Schematic representation of a 5.8-kb human WIPI-1 promoter fragment. Using MatInspector, 4 putative PU.1 binding sites (circles) are found in the human WIPI-1 promoter. (b) In vivo binding of PU.1 to the 4 binding sites was shown by ChIP in NB4 cells using antibodies against PU.1. Antibodies against acetyl-histone H3 and IgG served as positive and negative controls, respectively. GAPDH amplification was shown as a negative control for the different pull-downs. (c) Upper panel: WIPI-1 mRNA expression was measured in NB4 shPU.1 knockdown cells upon ATRA treatment at day 4. Lower panel: PU.1 western blot analysis of NB4 SHC002 control and PU.1 knockdown cells. Total protein expression was used as loading control. (d) NB4 cells, transduced with an inducible PU-1-ER expressing vector were treated with 4-OHT to induce PU.1 translocation to the nucleus. WIPI-1 mRNA levels were assessed by qPCR and normalized to the HMBS housekeeping gene. Results are given as n-fold regulation compared with untreated, control transduced NB4 pBabe cells. M.W.U, ***P<0.001. M.W.U, Mann–Whitney U-test
Figure 2 PU.1-dependent WIPI-1 regulation in NB4 APL cells. (a) Schematic representation of a 5.8-kb human WIPI-1 promoter fragment. Using MatInspector, 4 putative PU.1 binding sites (circles) are found in the human WIPI-1 promoter. (b) In vivo binding of PU.1 to the 4 binding sites was shown by ChIP in NB4 cells using antibodies against PU.1. Antibodies against acetyl-histone H3 and IgG served as positive and negative controls, respectively. GAPDH amplification was shown as a negative control for the different pull-downs. (c) Upper panel: WIPI-1 mRNA expression was measured in NB4 shPU.1 knockdown cells upon ATRA treatment at day 4. Lower panel: PU.1 western blot analysis of NB4 SHC002 control and PU.1 knockdown cells. Total protein expression was used as loading control. (d) NB4 cells, transduced with an inducible PU-1-ER expressing vector were treated with 4-OHT to induce PU.1 translocation to the nucleus. WIPI-1 mRNA levels were assessed by qPCR and normalized to the HMBS housekeeping gene. Results are given as n-fold regulation compared with untreated, control transduced NB4 pBabe cells. M.W.U, ***P<0.001. M.W.U, Mann–Whitney U-testCC BY-NC-SA · PubMed Central
Figure 3 Impaired neutrophil differentiation in NB4 WIPI-1, WIPI-2, PI3KC3 but not in BECN1 knockdown cells. (a) SHC002, shWIPI-1, shWIPI-2, shPI3KC3 and shBECN1 expressing NB4 cells were differentiated for 4 days and knockdown efficiency was measured by qPCR. (b) Neutrophil differentiation was assessed by measuring CD11b surface expression with FACS analysis, a representative CD11b histogram is shown. (c) Bar graphs of the median fluorescence intensity of three independent experiments are shown. (d) CEBPE mRNA expression was measured by qPCR in SHC002, shWIPI-1, shWIPI-2, shPI3KC3 and shBECN1 expressing NB4 cells upon 1 μM ATRA treatment after 4 days. (e) NBT assay was performed at day 4 in SHC, shWIPI-1, shWIPI-2, shPI3KC3 and shBECN1 NB4 APL cells and NBT-positive cells were quantified from two independent experiments done in duplicate. (f) Relative transcript abundance for WIPI-1 and WIPI-2 was calculated as described in.12 M.W.U, *P<0.05. **P<0.01, ***P<0.001. M.W.U, Mann–Whitney U-test
Figure 3 Impaired neutrophil differentiation in NB4 WIPI-1, WIPI-2, PI3KC3 but not in BECN1 knockdown cells. (a) SHC002, shWIPI-1, shWIPI-2, shPI3KC3 and shBECN1 expressing NB4 cells were differentiated for 4 days and knockdown efficiency was measured by qPCR. (b) Neutrophil differentiation was assessed by measuring CD11b surface expression with FACS analysis, a representative CD11b histogram is shown. (c) Bar graphs of the median fluorescence intensity of three independent experiments are shown. (d) CEBPE mRNA expression was measured by qPCR in SHC002, shWIPI-1, shWIPI-2, shPI3KC3 and shBECN1 expressing NB4 cells upon 1 μM ATRA treatment after 4 days. (e) NBT assay was performed at day 4 in SHC, shWIPI-1, shWIPI-2, shPI3KC3 and shBECN1 NB4 APL cells and NBT-positive cells were quantified from two independent experiments done in duplicate. (f) Relative transcript abundance for WIPI-1 and WIPI-2 was calculated as described in.12 M.W.U, *P<0.05. **P<0.01, ***P<0.001. M.W.U, Mann–Whitney U-testCC BY-NC-SA · PubMed Central
Figure 4 Attenuated differentiation of WIPI-1 and WIPI-2 knockdown APL cells is in part due to impaired autophagy. (a) Autophagic flux was measured in SHC002 and shWIPI-1 or shWIPI-2 expressing NB4 cells upon 1 μM ATRA treatment at day 4 in the presence or absence of bafilomycin A1 by measuring endogenous LC3-II protein levels on western blot and four independently performed experiments were quantified. GAPDH was used as loading control. (b) Stable EGFP-Cherry-LC3 expressing NB4 cells were transduced either with SHC002, shWIPI-1 or shWIPI-2 and cells were treated with 1 μM ATRA for 4 days. Bafilomycin A1 was added after 3 days. Representative pictures of EGFP-Cherry-LC3 puncta from three independently performed experiments are shown. (c) The rate of long-lived protein degradation was measured in control and ATRA-treated SHC002 control cells or WIPI-1, WIPI-2 knockdown cells, in the presence or absence of the lysosomal inhibitor bafilomycin A1.21 The degradation rate for long-lived proteins was calculated as the percentage of radioactivity in the TCA-soluble fraction relative to the total radioactivity in nonsoluble fractions. Further, values were subtracted from the corresponding sample treated with bafilomycin A1. (d) Long-lived protein degradation in NB4 SHC002 control and PU.1 knockdown cells. Analysis as in c. M.W.U, *P<0.05. **P<0.01. M.W.U, Mann–Whitney U-test
Figure 4 Attenuated differentiation of WIPI-1 and WIPI-2 knockdown APL cells is in part due to impaired autophagy. (a) Autophagic flux was measured in SHC002 and shWIPI-1 or shWIPI-2 expressing NB4 cells upon 1 μM ATRA treatment at day 4 in the presence or absence of bafilomycin A1 by measuring endogenous LC3-II protein levels on western blot and four independently performed experiments were quantified. GAPDH was used as loading control. (b) Stable EGFP-Cherry-LC3 expressing NB4 cells were transduced either with SHC002, shWIPI-1 or shWIPI-2 and cells were treated with 1 μM ATRA for 4 days. Bafilomycin A1 was added after 3 days. Representative pictures of EGFP-Cherry-LC3 puncta from three independently performed experiments are shown. (c) The rate of long-lived protein degradation was measured in control and ATRA-treated SHC002 control cells or WIPI-1, WIPI-2 knockdown cells, in the presence or absence of the lysosomal inhibitor bafilomycin A1.21 The degradation rate for long-lived proteins was calculated as the percentage of radioactivity in the TCA-soluble fraction relative to the total radioactivity in nonsoluble fractions. Further, values were subtracted from the corresponding sample treated with bafilomycin A1. (d) Long-lived protein degradation in NB4 SHC002 control and PU.1 knockdown cells. Analysis as in c. M.W.U, *P<0.05. **P<0.01. M.W.U, Mann–Whitney U-testCC BY-NC-SA · PubMed Central

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Metadata

Authors
Brigger, D., Proikas-Cezanne, T., & Tschan, M. P.
Venue
Cell Death & Disease
Publisher
Springer Science and Business Media LLC
Published
2014-07-03
Volume
5
Issue
7
Pages
e1315-e1315
ISSN
2041-4889
DOI
10.1038/cddis.2014.261
PMC
PMC4123064
Cited by
48
References
5
Language
en
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