Fig. 3: VSTM2L suppression promotes ferroptosis of PCa Cells.

A The cell morphology of 22Rv1 and Du145 cells with VSTM2L shRNA (shVSTM2L) or control shRNA (control) were observed by inverted light microscopy. Scale bars = 200 μm. B Representative transmission electron microscopy (TEM) images of 22Rv1 cells with control or shVSTM2L. Scale bars = 1 μm / 500 nm. C–F Representative images (C) and quantification analysis (D–F) of high resolution laser confocal microscopy for the morphology of mitochondria in DU145 cells transfected with control or shVSTM2L. Data shown as mean ± SD, n = 3 for each group, Scale bars = 10 μm. G–I The 22Rv1(G) and DU145 (H) cells with VSTM2L shRNA (shVSTM2L-1, shVSTM2L-2) or control shRNA (Control) were stained with propidium iodide (PI, 3 μg/mL) and analyzed by flow cytometry to evaluate the cell death rate. Data is shown as mean ± SD of n = 3 biological replicates. 22Rv1 (Control VS shVSTM2L-1 P = 6.1418E-7, Control VS shVSTM2L-2 P = 1.1758E-7), DU145 (Control VS shVSTM2L-2 P = 0.00002). J Lipid peroxidation was measured by C11-BODIPY (5 μM) in 22Rv1 (Control VS shVSTM2L-1 P = 0.000019, Control VS shVSTM2L-2 P = 5.9221E-8) and Du145 cells with control or VSTM2L shRNAs. Data shown as mean ± SD of n = 3 biological replicates. K The level of glutathione (GSH) in 22Rv1 and Du145 cells with control or VSTM2L shRNAs. Data shown as mean ± SD of n = 3 biological replicates. L Western blot analysis of GPX4 and VSTM2L protein levels in VSTM2L knockdown PCa cells or control cells. Protein levels were normalized to total GAPDH. P value was determined by unpaired two-tailed Student’s t-test without adjustments (D, E, F) and one-way ANOVA (I, J, K). Source data are provided as a Source Data file.