Fig. 5

Cleavages of RICTOR and RPTOR by caspases upon PI3K/mTOR dual inhibition. A Western blot analysis of RICTOR and RPTOR cleavages and the activation of CASP3 and CASP6 in NCI-H446 cells treated using GSK2126458 (200 and 1000 nM) for 12 h. Cleavage of PARP1 (c-PARP1) and a loading control GAPDH were monitored and compared. B NCI-H446 cells were first transfected with siRNAs targeting either or both CASP3 and CASP6 for 48 h. After another 12 h of GSK2126458 treatment (1.0 µM), cells were harvested and subjected to Western blot analysis using the indicated antibodies. C Quantification for the ratios of c-RICTOR, c-RPTOR, and c-PARP1 in Western blot experiments of (B) (error bars are mean ± SD from duplicated experiments; *p < 0.05; **p < 0.01; ***p < 0.001, t-test). D Effects of z-VAD-fmk on the GSK2126458-induced cleavage of RICTOR and RPTOR in NCI-H446 cells. Cells were treated using GSK2126458 with or without z-VAD-FMK (10 µM) for 3 or 12 h. E NCI-H446 cell lysates were incubated with various quantities of recombinant CASP3 or CASP6 (0.5, 1, 2, and 4 U) for 15, 30, 60, 120, and 180 min. Following the enzymatic reaction, cleavages of RICTOR and RPTOR were obtained using Western blot analysis. Levels of c-RICTOR and c-RPTOR were quantified and listed below the profiles. The molecular sizes of cleaved RICTOR and RPTOR in NCI-H446 cells treated with GSK2126458 (GSK) are presented for comparison