GLUT-specific modulation of signal transducers and apoptosis effectors: cytotoxicity of GLUT4 silencing is mediated by Mcl-1 suppression. (A) L363 cells were transduced with the indicated shRNAs, and cell lysates were prepared. Representative blots are shown. (B) KMS11 and JJN3 cells were transduced with control (C) or GLUT4-targeted shRNA and incubated for 4 days before lysate preparation. Representative blots are shown. (C) L363 cells were transduced with an empty vector control (EV), WT MCL1 (Mcl-1 WT), or ubiquitination-resistant MCL1 mutant (Mcl-1 5K). Stable cell lines were generated, and Mcl-1 expression was assessed by immunoblot analysis. Representative blot is shown. (D) L363 stable cell lines from panel C were transduced with control- or GLUT4-targeted shRNA and incubated for 3 days before immunoblot analysis of GLUT4, Mcl-1, and PARP. Representative blot is shown. (E) Cells from panel D were subjected to flow cytometric viability analysis via annexin V/DAPI staining. Data are normalized to control shRNA-expressing cells within each cell line. Data in panel E are mean ± SEM. (F) L363 cells were treated with DMSO (C) or 1μM PP242, the active site kinase inhibitor of mTOR, for the indicated lengths of time before protein extraction. Immunoblot analysis of phospho-4EBP1 and Mcl-1 levels is shown. GAPDH serves as a loading control. Representative blot is shown (n = 2). (G) Cells were transduced with control or GLUT4-targeted shRNA and incubated 4 days before RNA extraction, and real-time RT-PCR analysis of Mcl-1 mRNA expression was performed. Relative quantities are shown and normalized to control shRNA-expressing cells. (G) Data are mean ± SEM. (A-E,G) n ≥ 3. *P < .05. **P < .01. ***P < .005.