Fig 2: Loss of mTORC2 activity ablates IGF1-induced SGK3 activity.Wild-type and SIN1 knock-out HEK293 cells were serum-starved overnight prior to 50 ng/ml IGF1 stimulation for 15 min. Endogenous SGK3 was immunoprecipitated from the lysates and SGK3 kinase activity was assessed by measuring phosphorylation of the Crosstide substrate peptide in the presence of 0.1 mM 32PγATP in a 30 min reaction. Both immunoprecipitates and lysates were subjected to western blotting with the indicated antibodies.

Fig 3: Overexpression of Sin1 Reinforces Osteosarcoma Cell Invasion(A and B) Left: cell migration and invasion were detected by a Transwell chamber assay with or without Matrigel in MG63 cells (A) and U2OS cells (B) after SIN1 cDNA transfection and NC treatment. Right: quantitative results are illustrated for the left image. Control, pcDNA3.1; MG63, 1.5 μM NC; U2OS, 4 μM NC; Both, SIN1 cDNA+NC. (C) Top: the expression of SIN1 was detected by western blotting in osteosarcoma cells with SIN1 cDNA transfection and NC treatment. Bottom: quantitative results are illustrated for the top image. *p < 0.01 compared with control; #p < 0.05 compared with NC treatment alone or Sin1 cDNA transfection alone.

Fig 4: Effects of SIN1 Overexpression on Cell Growth Apoptosis and Migration(A) The effect of SIN1 overexpression in combination with NC treatment on osteosarcoma cell growth by MTT assay. Control, pcDNA3.1; MG63, 1.5 μM NC; U2OS, 4 μM NC; Both, Sin1 cDNA+NC. (B) Cell apoptosis in MG63 cells and U2OS cells was detected by flow cytometry after Sin1 cDNA transfection and NC treatment. (C) Left: cell migration was measured by a wound-healing assay after SIN1 cDNA transfection and NC treatment. Right: quantitative results are illustrated for the left image. *p < 0.01 compared with control; #p < 0.05 compared with NC treatment alone or SIN1 cDNA transfection alone.

Fig 5: NC Inhibits SIN1 Expression(A) The expression of SIN1 was measured by western blotting in MG63 cells and U2OS cells after NC treatment. (B) Quantitative results are illustrated for (A). *p < 0.01 compared with control (DMSO treatment group).