Fig 1: HRD1 interacts with CPT2 and decreases its protein stability. (A) CPT2 mRNA was detected in HRD1 knockdown cells by qRT–PCR. *P < 0.05, **P < 0.01, ns = not significant, ANOVA test. Data were expressed as mean ± SEM (n = 3 per group). (B) CPT1A and CPT2 protein expression was detected in HRD1 knockdown cells by western blot. (C) Cell lysates of MDA‐MB‐231 cells were immunoprecipitated with an IgG or HRD1 antibodies, and immunoblot assays were performed using HRD1, CPT2, or CPT1A antibody. (D, E) HEK 293T cells were transfected for 24 h with plasmids encoding either Flag‐CPT2 or Myc‐HRD1 alone or in combination. Cell lysates were immunoprecipitated with an anti‐Flag and anti‐Myc antibodies, and immunoblot assays were performed using anti‐Myc or anti‐Flag antibody. (F) HRD1 knockdown and shCon cells were treated with 40 μm cycloheximide for the indicated times. Cell lysates were immunoblotted with HRD1 and CPT2 antibodies. Relative levels of CPT2 were quantified by imagej. *P < 0.05, **P < 0.01, unpaired two‐tailed Student's t‐test. Data were expressed as mean ± SEM (n = 3 per group). (G) HEK 293T cells were transfected with Flag‐CPT2 alone or together with increasing doses (0.5, 1.0, and 1.5 μg) of Myc‐HRD1 for 24 h, and treated with or without 10 μm MG132 for 6 h. The expression of transfected CPT2 and HRD1 was confirmed by immunoblotting with anti‐Flag and anti‐Myc antibody. (H) Representative IHC staining of HRD1 and CPT2 in TNBC tissues. Scale bars, 50 μm. (I) Correlation analysis between HRD1 and CPT2 based on H‐Score. R represents Pearson correlation coefficient.
Fig 2: HRD1 knockdown promotes the proliferation of TNBC cells in vitro under glutamine‐deficient conditions. (A) Cells were cultured in 96‐well plates under normal culture condition, and cell viability was determined by CCK8 assay. ANOVA test. (B) Cells were cultured in 6‐well plates under normal culture condition for 14 days. Then, the number of colonies was calculated and indicated by the histogram. ANOVA test. (C, E) Cells were cultured in 96‐well plates under glucose‐deficient and glutamine‐deficient condition, respectively, and cell viability was determined by CCK8 assay. **P < 0.01, ANOVA test. (D, F) Cells were cultured in 6‐well plates under glucose‐deficient and glutamine‐deficient condition for 14 days, respectively. Then, the number of colonies was calculated and indicated by the histogram. **P < 0.01, ANOVA test. (G, H) HRD1 protein expression under glutamine deficiency condition in both time‐ and concentration‐dependent cases was analyzed by western blot. Data were expressed as mean ± SEM (n = 3 per group).
Fig 3: HRD1 targets Vimentin for polyubiquitination-mediated degradation. a Immunoblotting showing the expression of N-Cadherin and Vimentin in control cells and HRD1-overexpressing MDA-MB-231 and BT549 cell clones (#1-#3) with ß-actin as a loading control. b Agarose gel electrophoresis of RT-PCR products showing the mRNA expression of HRD1 and Vimentin in control and HRD1-overexpressing MDA-MB-231 and BT549 cells. Transcript of ß-actin was used as a loading control. c Vimentin mRNA level in MDA-MB-231 and BT549 cells before and after HRD1 overexpression, examined with real-time quantitative RT-PCR. ß-actin was used as an internal control. d Stability tests of Vimentin in control and HRD1-overexpressing MDA-MB-231 cells treated with 50 µg/ml of cycloheximide (CHX) for the indicated times. ß-actin was used as a loading control. e Immunoblotting showing the expression of Vimentin in control and HRD1-overexpression MDA-MB-231 and BT549 cells pretreated with MG132 (10 µM) or Bafilomycin A1 (BafA1, 100 nM) for 6 h. ß-actin was used as a loading control. f Endogenous interaction between HRD1 and Vimentin detected by in vitro co-immunoprecipitation assay in HEK293 cells cotransfected with Myc-HRD1 and Flag-Vimentin plasmids upon MG132 (10 µM) treatment. Whole-cell lysates were immunoprecipitated with anti-Myc antibody, then immunoblotted and hybridized with the indicated antibodies. g Immunofluorescence staining showing a co-localization of HRD1 and Vimentin in the cytoplasm of MDA-MB-231 cells transfected with Myc-HRD1 plasmids upon MG132 (10 µM) treatment. Fluorophore-conjugated secondary antibodies were used as green indicating HRD1 and red indicating Vimentin. DAPI was used for staining the nucleus. Images were taken with a laser scanning confocal microscope. Scale bar = 20 µm. h Ubiquitination of Vimentin in MDA-MB-231 cells transfected with HA-ubiquitin, Myc-HRD1 or Flag-Vimentin plasmids upon MG132 (10 µM) treatment. Whole-cell lysates were immunoprecipitated with anti-Flag antibody, then immunoblotted and hybridized with the indicated antibodies. i Whole-cell lysates of HEK293 cells cotransfected with Flag-Vimentin and Myc-HRD1 or Myc-HRD1 (C291S) plasmids upon MG132 (10 µM) treatment were immunoprecipitated with anti-Flag antibody, showing the interaction between Vimentin and wild-type HRD1 or C291S mutant. j Immunoblotting showing the expression of Vimentin in MDA-MB-231 and BT549 cells before and after the overexpression of wild-type HRD1 or C291S mutant. k Stability tests of Vimentin in MDA-MB-231 cells overexpressing wild-type HRD1 or C291 mutant, treated with CHX (50 µg/ml) for the indicated times. l In vitro ubiquitination assay of Vimentin similar to Fig. 3h, with the addition of HRD1 (C291S) overexpression
Fig 4: Expression profile of HRD1 in different subtypes of breast cancer. a Expression of HRD1 at the protein and mRNA level in luminal (T47D, MCF-7, BT474) and basal-like (MDA-MB-231, BT549) breast cancer cell lines, examined by immunoblotting and RT-PCR with ß-actin as a loading control. b RT-qPCR results of HRD1 expression in five kinds of breast cancer cells. ß-actin was used as an internal control. c Scatter plots of HRD1 expression in 52 breast cancer cell lines from public microarray dataset (GSE41313). Luminal N = 29, TNBC N = 23. d Representative immunohistochemical staining of HRD1 protein expression in luminal breast cancer and TNBC tissues. Scale bar = 50 µm. e RT-qPCR results of HRD1 mRNA level in FFPE luminal (N = 5) and TNBC (N = 5) tissues. ß-actin was used as an internal control. f Violin plots demonstrating the differential expression of HRD1 across the breast cancer subtypes, sampling from The Cancer Genome Atlas (TCGA-BRCA). LA (Luminal A) N = 486, LB (Luminal B) N = 79, HER2 N = 30, TNBC N = 123. One-way ANOVA was used for statistical analysis. g, h Box plots showing HRD1 expression in patients with luminal breast cancer or TNBC from public microarray datasets (GSE1456 and GSE5460). Luminal N = 62/76, TNBC N = 25/38. i Kaplan-Meier analysis showing the influence of HRD1 expression on relapse-free survival across the breast cancer subtypes from multiple public microarray datasets collected and organized by KM plotter. Data were represented as means ± S.D. of at least three independent experiments
Fig 5: Correlation between HRD1 and Vimentin protein expression in breast cancer. a list of the top 6 HRD1-binding protein candidates in MDA-MB-231 cells, analyzed by liquid chromatography-mass spectrometry assay. iBAQ = intensity-based absolute quantification. b Heatmap showing the protein expression of HRD1 and Vimentin assessed with mass spectrometry in breast cancer samples (N = 67) from the Clinical Proteomic Tumor Analysis Consortium (CPTAC). Color key = protein expression Z-score. c Correlation between HRD1 and Vimentin protein expression in breast cancer samples (N = 67) from CPTAC, determined by Pearson’s correlation coefficient. d Representative immunohistochemical staining of HRD1 and Vimentin protein expression in both luminal subtype and TNBC tissues. Scale bar = 50 µm. e Stacked bar charts exhibiting the distribution of breast cancer samples (N = 60) with different expression levels of HRD1 and Vimentin evaluated by the IHC score. 0 (-) and 1 (+) were regarded as low expression, while 2 (++) and 3 (+++) were regarded as high expression. Luminal N = 30, TNBC N = 30. Fisher’s exact test was performed for statistical analysis. f Paired line plots showing the IHC score of HRD1 and Vimentin in matched breast cancer tissue sections (N = 60). P value was calculated as Pearson’s correlation coefficient, showing a reverse correlation of HRD1 and Vimentin
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