Fig. 8: Gαi3 is important for Akt-mTOR activation in in bladder cancer cells.

The priBlCa-1 primary bladder cancer cells with Gαi3-specific lentiviral shRNA (shGαi3-S1, shGαi3-S2, or shGαi3-S3, representing different sequences), a scramble control shRNA (“shC”), the Cas9-expressing construct plus the CRISPR/Cas9-Gαi3-KO-puro construct (“koGαi3-C1” and “koGαi3-C2”, two stable colonies), the CRISPR/Cas9 empty vector (“koC”), a lentiviral Gαi3-overexpressing construct (“oeGαi3-stb slc1” and “oeGαi3-stb slc2,” representing two stable cell lines) or an empty vector (“Vec”) were cultured and expression of listed proteins was shown (A–C). shGαi3-S3-expressing priBlCa-1 cells were stably transduced with or without a constitutively-active S473D mutant Akt1 (caAkt1). The expression levels of the specified proteins were shown (D). These transduced cells were then cultured for the indicated durations, and assays were conducted to evaluate cell proliferation using EdU-nuclei staining (E), migration using the “Transwell” (F), and apoptosis using TUNEL-nuclei staining (G). Data were presented as mean ± standard deviation (SD). n = 5 stands for five biological repeats. Statistical significance was indicated by *P < 0.05 compared to the “shC”/“koC”/“Vec” cells. ^#P < 0.05. Each experiment was repeated five times, yielding consistent results. Scale bar = 100 μm.