FIG 5.

Inhibition of SCF-FBXW7 by knocking down Cul-1 and Skp1 (A) or FBXW7 (B and C) or by knocking out FBXW7 (D and E) rescues Mcl-1 reduction induced by TORKinibs (A to D) or rictor silencing (E), whereas TORKinibs decrease the levels of other known FBXW7 substrates (F). (A to C) The indicated cell lines were transfected with control (Ctrl) or other indicated siRNAs, and after 48 h, they were exposed to the indicated TORKinibs (100 nM) for an additional 4 h. (D) WT and FBXW7-KO HCT116 cells were treated with the indicated TORKinibs at 100 nM for 3 h. (E) WT and FBXW7-KO HCT116 cells were transfected with the indicated siRNAs for 48 h. After these treatments, the cells were harvested for preparation of whole-cell protein lysates and subsequent Western blotting. FBXW7 knockdown efficiency in panel B was evaluated by RT-PCR. SE, short exposure. (F) A549 cells were treated for 8 h with the different TORKinibs indicated at 100 nM and then harvested for preparation of whole-cell protein lysates and subsequent Western blotting.