Fig. 8: VSTM2L plays oncogenic roles in PCa by inhibiting VDAC1 oligomerization.
A VSTM2L knockdown DU145 cells cultured with or without VBIT4 (5 μM) for 48 h were harvested for the subsequent cross-link assay, then subject to western blot to assess VDAC1 oligomeric status. The samples derived from the same experiment and that gels/blots were processed in parallel. B–D VSTM2L suppressed DU145 cells were cultured with or without VBIT4 (5 μM) for the indicated time, then the cell viability (n = 5 biological replicates) (B) and colony formation ability (n = 4 biological replicates) (C, D) were analyzed by CCK8 assay and Crystal Violet Aqueous Solution staining, respectively. Data shown as mean ± SD. ns not significant. E, F Wound healing analysis of VSTM2L inhibited DU145 cells in the absence or presence of VBIT4 (5 μM). Representative images (E) and quantification (F) are shown as indicated. Data shown as mean ± SD of n = 3 biological replicates. ns not significant, Scale bars = 200 μm. G, H Representative pictures (G) and quantification analysis (H) of migration assays in VSTM2L knockdown DU145 cells cultured with or without VBIT4 (5 μM). Data shown as mean ± SD of n = 3 biological replicates. ns not significant, scale bars = 250 μm. I A schematic diagram of the in vivo experimental process. 3.5 × 106 DU145 shVSTM2L cells and control cells were inoculated trans-subcutaneous into nude mice. The mice were administrated with VBIT4 (20 mg/kg) by gavage on alternate days until the tumor tissues were isolated on 31th days post-inoculation. J, K General view of tumor weight of each indicated groups at the endpoint. Data shown as mean ± SD, n = 5 tumors, ns not significant. L Tumor growth curves were shown. Data shown as mean ± SD, n = 5 tumors, ns not significant. M–Q Immunohistochemistry (IHC) and hematoxylin and eosin (H & E) staining for Ki-67 (M, N), VSTM2L (M, O), GPX4 (M, P) and C-Caspase3 (M, Q) were performed in isolated tumor tissues. Data shown as mean ± SD, n = 5 tumors and each tumor slice was randomly selected 5 magnification fields, Scale bars =20 μm. P value was determined by two-way ANOVA (B, D, F, H, L) and unpaired two-tailed Student’s t-test without adjustments (K, N–Q). Source data are provided as a Source Data file.
