Fig 2: Activation of SH3BGRL-PIK3C3/ATG12 autophagy axis in breast cancer patients. (A). Representative IHC staining of SH3BGRL, ATG12, PIK3C3 and LC3B expressions in 25 primary breast cancer specimens along with the matched adjacent normal tissues. Scale bar: 50 µm. (B,C) Statistical analysis of ATG12 (B) and PIK3C3 (C) in tissues of (A). (D) Correlation analysis of SH3BGRL protein level to that of ATG12 (r = 0.42; P = 0.037) or PIK3C3 (r = 0.49; P = 0.01) in breast cancer tissues in (A). (E) Immunoblots of the indicated proteins in 14 fresh breast cancer specimens. ACTB was used as a loading control. (F) Heatmap of protein expression of SH3BGRL, PIK3C3, ATG12, SQSTM1, and LC3B protein band intensity in (E), which was quantified and analyzed with densitometry and Image J software. (G) Correlation analysis of SH3BGRL protein level with that of PIK3C3, ATG12, SQSTM1 or LC3B in (F). (H) Marginal elevation of PIK3C3 mRNA expression in breast cancer tissues, compared with normal breast tissues (T = 1092, N = 111;T:N = 0.83, P < 0.0001; TCGA) and ATG12 mRNA (T = 1092, N = 111;T:N = 1.02, P < 0.0001; TCGA). (I) Kaplan–Meier relapse-free survival curves of breast cancer patients based on mRNA of ATG12 (n = 3951; P = 1.1e-11) or PIK3C3 (n = 3951; P = 1.5e-4). (J) Schematic mechanism of SH3BGRL-PIK3C3/ATG12 autophagy on doxorubicin resistance in breast cancer. All statistical analyses are shown as ****P < 0.0001.

Fig 3: Pathway by which RCE-4 inhibits the formation of the Bcl-2–Beclin 1 complex. (A) Protein expression levels in Ca Ski cells treated with RCE-4 at different time points were analyzed by western blotting. β-actin was used as the loading control. (B) The content of Beclin 1–HMGB-1 complex was detected by Co-IP assay. Data are presented as the mean ± standard deviation of three independent experiments. *** p < 0.001 vs. Control. (C) The content of Beclin 1–ATG 14–Vps34–Vps15 complex was detected by Co-IP assay. Data are presented as the mean ± standard deviation of three independent experiments. *** p < 0.001 vs. Control. (D) The content of Beclin 1–Vps34–Vps15–UVRAG complex was detected by Co-IP assay. Data are presented as the mean ± standard deviation of three independent experiments. ** p < 0.01, *** p < 0.001 vs. Control. (E) The content of Beclin 1–Vps34–Vps15–UVRAG–Rubicon complex was detected by Co-IP assay. Data are presented as the mean ± standard deviation of three independent experiments. *** p < 0.001 vs. Control.

Fig 4: WASH mediates RNF2 recruitment for AMBRA1 degradation. (A) WASH interacts with RNF2 in yeast two-hybrid assays. Yeast strain AH109 was transfected with BD-fused WASH and AD-fused RNF2 and plated on the indicated medium for 3 d. TP53 with large T antigen or a known WASH interactor Bloc1s2 (data not shown) was introduced as positive controls. (B) WASH directly interacts with RNF2. Recombinant WASH (rWASH) and GST-RNF2 were subjected to GST pull-down assays. (C) WASH enhances the interaction between RNF2 and AMBRA1. GST-RNF2 or GST was incubated with recombinant AMBRA1 with or without WASH, followed by GST pull-down assays. (D) RNF2 associates with the components of the BECN1-PIK3C3 complex. Cell lysates from MEFs treated with or without EBSS for 2 h were immunoprecipitated with anti-RNF2 antibody. (E, F) WASH promotes degradation of AMBRA1. MEFs transfected with MYC-AMBRA1 and increasing amounts of FLAG-WASH were harvested 24 h post-transfection for immunoblotting with the indicated antibodies (E). mRNAs were extracted and subjected to RT-PCR analysis with primers specific for exogenous plasmids (F). GFP served as a transfection control. (G) K45R-AMBRA1 mutation abolishes WASH-augmented degradation of AMBRA1. HA-tagged WASH was transfected into AMBRA1-depleted MEFs rescued with WT- or K45R-AMBRA1 for 24 h. Cells were starved in EBSS for 4 h, and harvested for immunoblotting with the indicated antibodies. (H) WASH deficiency prevents degradation of AMBRA1. WASH+/+ or WASH−/− MEFs treated with 20 μg/ml CHX plus EBSS for the indicated times were harvested for immunoblotting (left panel). Blot band intensity ratios of AMBRA1/ACTB were calculated and shown as means ± SD (right panel). (I) AMBRA1 associates with RNF2 dependently of WASH. WASH+/+ or WASH−/− MEFs were treated with or without EBSS for 2 h, and cell lysates were immunoprecipitated with anti-RNF2 antibody. (J) WASH deficiency impairs polyubiquitination of AMBRA1. WASH+/+ or WASH−/− MEFs were starved in EBSS for the indicated times, followed by IP with antibody against AMBRA1 after pretreatment with 10 μM MG132 for 6 h. Immunoprecipitates were immunoblotted with the indicated antibodies. (K) WASH promotes polyubiquitination of AMBRA1 in vitro. Recombinant RNF2 was incubated with purified FLAG-AMBRA1, E1 (UBA1), E2 (UBE2D3) and ubiquitin mutants with or without recombinant WASH at 30 °C for 1 h, followed by IP with antibody against FLAG and immunoblotting with the indicated antibodies. Experiments were repeated three times with similar results.

Fig 5: PIK3C3 and ATG12 contribute to SH3BGRL-mediated Dox resistance. (A,B) Immunoblots of PIK3C3 (A) and ATG12 (B) in indicated cells. ACTB served as the loading control. (C) Flow cytometry analysis of the indicated MCF-7 series cells. 10 nM or 20 nM Dox was used to treat MCF-7 or MDA-MB-453 cells for 12 h or 24 h, respectively. The left panel shows the staining of ANXA5-FITC and PI. The right panel is the quantification of apoptotic cells. (D) Immunoblots of the indicated autophagy markers in MCF-7-SH3BGRL overexpression cells with either ATG12 or PIK3C3 knockdown, or in MDA-MB-453-SH3BGRL knockdown cells with either ATG12 or PIK3C3 forced expression. (E) Immunoblots of SQSTM1 and the cleaved CASP3 in the above cells in (C). MCF-7 cells were treated with 10 nM Dox for 12 h, and MDA-MB-453 cells with 20 nM Dox for 24 h. (F) Representative images of colonies formed by indicated cells with10 nM or 20 nM Dox treatment. The right panel shows the quantification of colony formation. Colonies with more than 60 cells were scored. (G) Statistical SQSTM1 and the cleaved CASP3 expression by three independent experiments. 10 nM Dox was used for MCF-7 cells, and 20 nM for MDA-MB-453 cells. (H) Cell viability analysis of indicated cells under treatment with Dox at various concentrations, combined with or without CQ for 24 h. (I) IC50 of the indicated cells under treatment as in (D). (J) Representative images of soft agar colonies of the indicated cells after treatments with10 nM or 20 nM Dox treatment for 48 h (Left panel). Quantification of soft agar colony is shown in the right panel. Colonies (larger than 0.1 mm diameter) were quantified after 18 days of culture. Significant differences are shown as *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, and n.s means no significance.