Fig 1: For figure legend, see page 1093. Figure 9 (See previous page). VDAC2 interacts with BECN1. (A) 293T cells were transiently transfected with pFlag-Vdac2 or both pBecn1-EGFP and p3xFlag-Vdac2, and after transfection for 48 h, the whole cell lysate was extracted for coimmunoprecipitation with anti-FLAG (mouse IgG for control), anti-BECN1 or anti-GFP (rabbit IgG for control). Anti-BECN1, anti-GFP or anti-FLAG antibody (input) was used for western blotting. (B) Colocalization analysis of VDAC2 and BECN1. HeLa cells were transiently cotransfected with pBecn1-EGFP and pVdac2-DsRed, followed by confocal microscopy. Colocalizing structures are indicated in yellow (merge), and the white arrows show the overlapping signals in the cells. HeLa cells were transiently cotransfected with both pBecn1-EGFP and pMito-DsRed or both pVdac2-EGFP and pMito-DsRed to show localization of BECN1 or VDAC2 as controls. (C) Schematic diagram of the mouse BECN1 wild type and various deletion and truncated mutants. The conserved domains (BH3, CCD and ECD) are indicated in boxes. (D) Coimmunoprecipitation between VDAC2 and deletion mutants of BECN1 domains. p3xFlag-Vdac2 was transiently cotransfected with pMyc-Bech1BH3Δ, pMyc-Becn1CCDΔ, or pMyc-Becn1ECDΔ in 293T cells. Cell lysates were examined by western blotting using the anti-MYC or anti-FLAG antibody (input). For coimmunoprecipitation, the lysates were immunoprecipitated with anti-FLAG, followed by immunoblotting with the anti-MYC antibody. All the domain-deletion mutants of BECN1 interact with VDAC2. (E) Colocalization analysis between VDAC2 and deletion mutants of BECN1 domains. HeLa cells were transiently cotransfected with pVdac2-DsRed and pBecn1BH3Δ-EGFP, pVdac2-DsRed and pBecn1CCDΔ-EGFP, or pVdac2-DsRed and pBecn1ECDΔ-EGFP. Colocalizing structures are indicated in yellow (merge). (F) Coimmunoprecipitation analysis shows that VDAC2 interacts with the N terminus of BECN1. 3xFLAG-VDAC2 was cotransfected with pMyc-Becn11 to 138, pMyc-Becn1139 to 353, pMyc-Becn1354 to 448, pMyc-Becn11 to 353, pMyc-Becn1139 to 448, pMyc-Becn11 to 105, or pMyc-Becn1106 to 448 into 293T cells. The cell lysates were examined by western blotting using the anti-MYC or anti-FLAG antibody (input). For coimmunoprecipitation, the lysates were immunoprecipitated with the anti-FLAG antibody, followed by immunoblotting with the anti-MYC antibody. FLAG-VDAC2 can interact with MYC-BECN11 to 138, MYC-BECN11 to 353, and MYC-BECN11 to 105. (G) Colocalization analysis of VDAC2 with BECN1 truncated mutants. VDAC2-DsRed was transiently cotransfected with BECN11 to 353-EGFP, BECN1139 to 448-EGFP, BECN11 to 105-EGFP or BECN1106 to 448-EGFP in HeLa cells. Colocalizing structures are indicated in yellow (merge). Arrows indicate that the signals overlapped between BECN1 and VDAC2. The nuclei were stained with Hoechst reagent. Scale bar: 10 μm.
Fig 2: VDAC2 promotes BECN1 interaction with BCL2L1. (A) VDAC2 interaction with BCL2L1 but not BCL2. 3xFLAG-VDAC2 was transiently cotransfected with pMyc-Bcl2 or pMyc-Bcl2l1 into 293T cells. The lysates were immunoprecipitated with the anti-MYC or anti-FLAG antibody, followed by immunoblotting with the anti-FLAG or anti-MYC antibody, respectively. The whole cell lysates were examined by western blotting using the anti-MYC or anti-FLAG antibody (input). (B) Colocalization of VDAC2, BCL2L1 and BECN1. HeLa cells were cotransfected with pVdac2-DsRed, pBecn1-EGFP and pMyc-Bcl2l1, followed by confocal microscopy. The MYC-BCL2L1 protein was detected by immunoblotting with the anti-MYC antibody (blue). Merged yellow signals indicate colocalizing structures (white arrows). Scale bar: 10 μm. (C to E) Coimmunoprecipitation analysis of interaction among VDAC2, BECN1 and BCL2L1 in mouse ovary. (C) The mouse ovary lysates were immunoprecipitated with anti-VDAC2, followed by immunoblotting with the anti-VDAC2, anti-BECN1 or anti-BCL2L1 antibody. (D) The mouse ovary lysates were immunoprecipitated with anti-BECN1, followed by immunoblotting with the anti-VDAC2, anti-BECN1 or anti-BCL2L1 antibody. (E) The mouse ovary lysates were immunoprecipitated with anti-BCL2L1, followed by immunoblotting with the anti-VDAC2, anti-BECN1, or anti-BCL2L1 antibody. (F) VDAC2 promotes the BECN1 interaction with BCL2L1. pMyc-Vdac2 (0, 0.3, 0.6,and 1.2μg) was cotransfected with 1μg pFlag-Bcl2l1 and 1 μg pMyc-Becn1 into 293T cells. The cell lysates were immunoprecipitated with anti-Flag followed by immunoblotting with anti-MYC antibody. The whole cell lysates were examined by western blotting using the anti-MYC or anti-FLAG antibody (input). (G) pMyc-Vdac2 (0, 0.3, 0.6, and 1.2 μg) cotransfected with pFlag-Bcl2l1 (1 μg) and the BECN1 N-terminal deletion mutant (pMyc-Becn1106 to 448) (1 μg) into 293T cells. Coimmunoprecipitation was performed as in panel (F). VDAC2 promotion to the interaction between FLAG-BCL2L1 and the BECN1 N-terminal deletion mutant was not detected.
Fig 3: SH3BP5-AS1 exerts its promoting impacts on BC cell ferroptosis via VDAC2. (A) qPCR was applied to examine VDAC2 expression in the cells with transfection of sh-VDAC2-1/2/3. (B) Ferrous iron content in BC cells was detected with a microplate reader after the transfection of indicated plasmids including pcDNA3.1, pcDNA3.1-SH3BP5-AS1, and pcDNA3.1-SH3BP5-AS1 + sh-VDAC2. (C) Lipid ROS was measured in the cells with transfection of indicated plasmids. **p < 0.01.
Fig 4: Ceramide-induced apoptosis critically relies on Glu84 in VDAC2. a WT and ΔVDAC1/2 HCT116 cells stably transduced with HA-tagged VDAC1, VDAC1E73Q, VDAC2, or VDAC2E84Q were transfected with empty vector (EV), Flag-tagged mitoCERT, or Flag-tagged mitoCERTΔSTART. At 24 h post transfection, cells were processed for immunoblotting with antibodies against PARP1, cleaved caspase-3 (Casp3), the Flag-epitope, the HA-epitope, and β-actin. FL full-length, CL cleaved. b Quantitative analysis of PARP1 cleavage in cells treated as in a. Data are means ± s.d.; n = 4; *p < 0.05, **p < 0.01 and ***p < 0.001 by two-tailed paired t-test. c Quantitative analysis of cleaved Casp3 in cells treated as in a. Data are means ± s.e.; n = 2. Source data
Fig 5: Ferlixit/erastin co-treatment leads to ferroptosis in COV318 cells. COV318 cells were treated with different concentrations (100 and 250 µM for 24 and 48 h) of ferlixit alone or in combination with 8 µM erastin for 8 h. Cell viability and mortality were measured using the MTT assay (A) and the PI flow cytometry assay (B), respectively. Results are shown as means ± SD of three independent experiments. *p-value <0.05, untreated vs treatment; ns, not significant. (C) Optical microscopy images showing the presence or absence of the ballooning phenotype in COV318 cells upon treatment with ferlixit and erastin alone or in combination. (D) Analysis of mitochondrial membrane potential, cytosolic and mitochondrial ROS production by TMRM, CM-H2DCFDA and MitoSOX Red flow cytometry assays in COV318 cells untreated, treated with ferlixit and erastin alone or in combination. (E) Western Blot of VDAC2, NCOA4, and FtH in COV318 cells upon administration of ferlixit and erastin alone or in combination. γ-TUB was used as a normalization control for protein quantification. (F) Mitochondrial ultrastructural images detected by TEM in COV318 cells upon treatment with ferlixit and ferlixit alone or in combination. Green arrows, intact mitochondria; red arrows, altered mitochondria; Nu, nucleus. All data are representative of three independent experiments.
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