Figure S6 from Genetic Silencing of AKT Induces Melanoma Cell Death Via Mtor Suppression

Supplementary Figure S6 shows that INY-03-041 leads to a substantial effect on melanoma cell proliferation but it is not as potent as siAKT1-3. A, Cell confluency assay of MTG001 and MTG004 cell lines treated with DMSO control, GDC0068 (1µM), INY-03-041 (1µM), GDC0941 (1µM), Lenalidomide (1µM), or siAKT1-3 (50nM). Treatment with INY-03-041 led to decreased cell proliferation (P < 0.0001 in each cell line), but did not lead to cell death, similar to siAKT1-3 in MTG001 and MTG004. Lenalidomide, a recruiter of the E3 ubiquitin ligase Cereblon, conjugated to GDC0068 to form INY-03-041, was used as a negative control for the off-target effects of INY-03-041. Error bars indicate standard error of the mean of triplicate wells. B, Immunoblotting of cell lysates treated with the DMSO control, MK2206 (2.5µM), GDC0068 (1µM), INY-03-041 (500nM), GSK0394 (3µM), GDC0941 (1µM), BYL719 (1µM), siAKT1-3 (50nM) at 22 h, and siAKT1-3 (50nM) for 24 h revealed complete knockdown of P-AKT (Ser473) and total AKT by siAKT1-3 at 24 h, as well as knockdown of P-PRAS40, P-S6 kinase, and P-4EBP1. INY-03-041 displayed a 77% reduction in total AKT compared to the 89% reduction by siAKT1-3. C, Time-course immunoblotting of cell lysates treated with varying concentrations of INY-03-041 demonstrated an incomplete total protein knockdown of individual AKT paralogs. D, Densitometry quantification of time-course immunoblotting found in C.