Fig. 6: TIMM13 is important for Akt-mTOR activation in primary human OS cells.

The primary OS cells, pOS-1, expressing the TIMM13 shRNA (“shTIMM13-S1”/“shTIMM13-S2”, with different sequences), the CRISPR/Cas9–TIMM13–KO construct (“koTIMM13”), or the scramble control shRNA plus the Cas9 empty vector (“shC+koC”), were established; Expression of listed proteins was shown (A). The primary pOS-1 cells, expressing the GV248-TIMM13-GFP-expressing construct (TIMM13OE-S1/S2, two stable selections) or the GV248 empty vector (“Vec”) were established, and expression of listed proteins was shown (B). The koTIMM13 pOS-1 cells were transduced with adenovirus-packed constitutively-active Akt1 (caAkt1, S473D), and stable cells were established. Expression of listed proteins was shown (C); cells were further cultivated under the complete medium for designated hours, and cell proliferation, migration, and apoptosis were examined by EdU staining (D), “Transwell” (E), and TUNEL staining (F) assays, respectively, with results quantified. TIMM13OE-S1 cells were treated with or without LY294002 (“LY”, 250 nM) for designated hours, expression of listed proteins was shown (G); cell proliferation (H) and migration (I) were tested. Error bars stand for mean ± standard deviation (SD, n = 5). *P < 0.05 vs. “shC+koC”/“Vec” cells. *P < 0.05 (C–I). Experiments in this figure were repeated five times. Scale bar = 100 μm.