FIG 4.

Inhibition of GSK3 with SB216763, CHIR99021, or siRNA rescues Mcl-1 degradation induced by TORKinibs (A to D), and GSK3 phosphorylation of Mcl-1 is critical for Mcl-1 reduction by TORKinibs (E and F). (A and B) A549 cells were pretreated with 10 μM SB216763 or CHIR99021 for 30 min and then cotreated with the indicated TORKinibs (100 nM for INK128, AZD8055, and Torin 1 and 1 μM for PP242) for an additional 10 h (A) or 6 h (B). (C) A549 cells were transfected with the indicated siRNAs for 48 h and then exposed to 100 nM INK128 for an additional 4 h. (D) A549 cells were treated with DMSO, 100 nM INK128, or INK128 plus 10 μM SB216763 or CHIR99021 for 6 h. The cells were then washed with PBS three times and refed with fresh medium containing 10 μg/ml CHX. At the indicated times, the cells were harvested for preparation of whole-cell protein lysates and subsequent Western blot analysis. Protein levels were quantified with NIH ImageJ software and normalized to tubulin. (E) A549 cells were exposed to 100 nM INK128 for different times as indicated and then harvested for preparation of whole-cell protein lysates and subsequent Western blot analysis. (F) 293T cells were transfected with plasmids carrying the indicated genes, and after 48 h, they were treated for an additional 10 h with 100 nM TORKinibs as indicated. After the aforementioned treatments, whole-cell protein lysates were prepared from these cells for Western blotting to detect the indicated proteins. Ctrl, control.