Fig 1: VDAC2 promotes the association of BAX and BAK with a VDAC complex. a Endogenous BAX and BAK associate with independent complexes in mitochondria. Mitochondria-enriched fractions from HeLa or HCT116 cells were solubilized in 1% digitonin prior to incubation with a control antibody or an antibody that binds inactive human BAK (7D10), prior to BN-PAGE and immunoblotting for BAK or BAX. * likely cross-reactivity of anti-rat secondary antibody with rat IgG used for gel-shift. Importantly, whilst all of the BAK:VDAC2 complex was gel-shifted by the BAK antibody, the BAX:VDAC2 complex was unaffected. b Mass spectrometry analysis of the native BAX complex. Mitochondria from MEFs expressing FLAG-BAXS184L or untagged BAXS184L were solubilized in 1% digitonin prior to anti-FLAG affinity purification and proteins identified by quantitative mass spectrometry analysis. Volcano plot illustrating the log2 protein ratios of proteins enriched in the native complex following quantitative pipeline analysis. Proteins were deemed differentially regulated if the log2 fold change in protein expression was greater than two-fold (red) or four-fold (green) and a –log10 p value = 1.3, equivalent to a p value = 0.05. c Mass spectrometry of the native BAK complex. Mitochondria from MEFs expressing FLAG-BAK or untagged BAK harvested and analyzed as in (b). d Deletion of VDAC2 impacts mitochondrial localization of BAX and BAK. Clonal populations of Bax-/- and Bak-/- MEFs with deleted Vdac1, Vdac2 or Vdac3 (denoted ?) were fractionated into cytosol and membrane and immunoblotted for BAX, BAK or TIM44 as a mitochondrial control. e VDAC2 plays the major role in BAX and BAK complex stability. Mitochondria isolated from clonal populations of Bax-/- and Bak-/- MEFs with deleted Vdac1, Vdac2 or Vdac3 were analyzed on BN-PAGE. Data are representative of two independent clones (see Supplementary Fig. 2e). Intermediate complexes indicated (arrows). f BAX-mediated apoptosis is impaired in the absence of VDAC2 and to a lesser extent by VDAC3. Polyclonal populations were treated with etoposide (10 µM) and cell death was assessed by PI uptake. Data are mean+/- SEM of three independent experiments. ***p < 0.001; **p < 0.01; n.s, not significant; based on unpaired Student’s t-test
Fig 2: The mitigating effects of P. histicolaon ferroptotic parameters(A) Perl’s staining of the gastric mucosa. Scale bar = 50 µm. (B) The iron content of the gastric mucosa. (C) Gastric mucosal MDA. The qRT-PCR analysis of iron homeostasis-related genes, HMOX-1, SLC39A14 and TFR1(D), pro-ferroptotic ACSL4 and COX-2 (E), and VDACs (F). Western blot analysis of ACSL4 (G), TFR1 (H), VDAC1 (I) and VDAC3 (J) in the gastric mucosa. Data are presented as the mean ± SD. (n = 7–8/group for qRT-PCR, n = 3/group for Western blot). Statistical significance by the one-way ANOVA. *P < 0.05, **P < 0.01
Fig 3: Silencing CAV2, PHLDA1, and VDAC3 separately significantly increases sensitivity to cisplatin. A–C qRT-PCR and Western blot showing CAV2, PHLDA1, and VDAC3 knockdown 48 and 72 h after transfection with CAV2-targeting, PHLDA1-targeting and VDAC3-targeting siRNAs. D: cytotoxicity curves of the LUAD cell lines A549, PC9 and ESCC cell line TE1 transfected with nontargeting (ctrl) or CAV2-targeting, PHLDA1-targeting and VDAC3-targeting siRNAs and treated for 48 h with different concentrations of cisplatin (0, 2.5, 5, 10, 20, 40 µM). E EdU for A549, PC9 and TE1 transfected with nontargeting (ctrl) or CAV2-targeting, PHLDA1-targeting and VDAC3-targeting siRNAs and treated for 48 h with cisplatin (10 µM)
Supplier Page from Proteintech Group Inc for VDAC3 antibody