Fig. 4: MTCH2 silencing causes extensive mitochondrial damage in primary human CRPC cells.

The primary pPC-1 cells were subjected to individual treatments with specific MTCH2 shRNAs (shMTCH2-S1, shMTCH2-S2, or shMTCH2-S3, each representing a unique sequence) or a control scramble non-sense shRNA (c-sh), stable cells were formed after puromycin selection. Expression levels of MTCH1 and MTCH2 were assessed using qPCR (A) and Western blotting (B) assays. An equal number of cells were cultured for designated time periods, and several key parameters were evaluated, including the mitochondrial complex I activity (C), ATP contents (D), and oxygen consumption rate (measured using the Seahorse assay, E), as well as the GSH/GSSH ratio (F, the left panel) and lipid peroxidation. (measured using the TBAR assay, F, the right panel). Mitochondrial depolarization was determined by JC-1 green monomer intensity (G), and the ROS levels were measured via quantifying CellROX and Mito-SOX fluorescence intensity (H, I). Additionally, the stable cells derived from other primary CRPC cells (pPC-2, pPC-3, and pPC-4) expressing either c-sh or shMTCH2-S1 were established. The mRNA expression levels of MTCH1 and MTCH2 in these cells were evaluated (J, K). Cells were cultured for the specific durations, and the mitochondrial complex I activity (L), ATP content (M), mitochondrial depolarization (tested via JC-1 green monomer intensity, N), and ROS production (measured by CellROX intensity, O) were tested similarly. The data are expressed as mean ± standard deviation (SD, n = 5). “Ctrl” denotes the parental control cells. Statistical significance is marked by *P < 0.05 when compared to “c-sh” cells. “n.s.” denotes non-statistically significant differences (P > 0.05). The experiments depicted in this figure were conducted five times (biological replicates), consistently producing similar results. The scale bar represents 100 μm.