Fig. 2

Pharmacological and genetic inhibition of GSK-3 suppresses 4EBP1, S6K, and S6RP phosphorylation in the mTORC1 downstream signaling cascade aberrantly activated in RCC cell lines. a The human RCC cell lines ACHN, Caki1, and A498 cells were treated with AR-A014418 (25 μM or 50 μM) for 24 h or 48 h, respectively. b ACHN cells were treated with SB-216763 (25 μM or 50 μM), and with TDZD8 (5 μM or 10 μM) for 24 h or 48 h, respectively. c ACHN cells were transfected with non-specific control siRNA (50 nM) as negative control (NC), GSK-3β-targeting (siGSK3β) siRNAs (25 nM or 50 nM) and GSK-3α-targeting (siGSK3α) siRNAs (50nM) for 48 h after seeding. d Human renal proximal tubular epithelial cell (HRPTEpC) and normal kidney tissues had slight expression of GSK-3 and the mTORC1 downstream substances (p4EBP1, pS6K and pS6RP). In contrast, GSK-3 and the mTORC1 downstream signaling pathway were strongly activated in RCC cells. In a–d, immunoblot analysis was performed after cell lysis. Data are representative of at least three separate experiments