Fig. 3: TIMM13 depletion provokes mitochondrial apoptosis cascade in OS cells.

The primary OS cells, pOS-1, expressing the TIMM13 shRNA (“shTIMM13-S1”/“shTIMM13-S2”, with different sequences), the CRISPR/Cas9–TIMM13–KO plasmid (“koTIMM13”), or the scramble control shRNA plus the Cas9 empty vector (“shC+koC”), were established and cultivated under the complete medium for designated hours, the relative caspase-3 activity (A) was examined; cell apoptosis was tested by TUNEL staining assay (B); cell death was quantified through Trypan blue staining assays (C). pOS-1 cells with shTIMM13-S1 or shC were treated with zVAD-fmk (40 μM), zDEVD-fmk (40 μM), NAC (400 μM) or ATP (1 mM), and cells were cultivated under the complete medium for designated hours; cell apoptosis and death were tested by TUNEL staining and Trypan blue staining assays (D, E). The primary OS cells, pOS-2, or the immortalized MG63 cells, with shTIMM13-S1 (“shTIMM13”) or the scramble control shRNA (“shC”), were cultivated under the complete medium for designated hours; The relative caspase-3 activity (F), cell apoptosis (by measuring TUNEL-positive nuclei percentage, G) and cell death (Trypan blue ratio, H) were tested. “Pare” stands for the parental control OS cells. Error bars stand for mean ± standard deviation (SD, n = 5). *P < 0.05 versus “Pare”/“shC” cells. ^#P < 0.05 versus “DMSO”/“PBS” (D, E). Experiments in this figure were repeated five times. Scale bar = 100 μm.