Figure 6.

mTOR signalling pathway is deeply involved in the regulation of P311 on VEGF expression in MSCs. (A–F) The activation of mTOR (P70S6K/p-P70S6K, 4EBP1/p-4EBP1, mTOR/p-mTOR, AKT/p-AKT, and PI3K/p-PI3K) signalling pathway in MSCs, MSCs^Ctrl and MSCs^P311 was detected using Western blot. Representative images for mTOR related signaling pathway were shown (left panel), and the ratio of phosphorylated to total proteins were statistically analysed (right panel). (G–K) The affection of mTOR related signalling pathway on P311-mediated up-regulating VEGF expression of MSCs was estimated by detecting VEGF extracellular secretion, intracellular expression of VEGF in MSCs^P311 with rapamycin (mTOR inhibitor), LY2584702 (P70S6K inhibitor), LY294002 (PI3K inhibitor), or control vehicle (DMSO) treatment. (G) The extracellular secretion of VEGF in MSCs^P311 with rapamycin, LY2584702, LY294002 or DMSO treatment was examined by ELISA assays. (H–K) The intracellular expression of VEGF in MSCs^P311 with rapamycin, LY2584702, LY294002, or DMSO treatment was detected by Western blot and confocal microscopy. (H, I) Representative immunoblotting images for VEGF were shown (left panel), and the mean gray value of VEGF was statistically analysed (right panel). (J, K) Representative fluorescent images for VEGF were shown (left panel), and the mean fluorescence intensity value of VEGF was statistically analysed (right panel). Scale bar: 100 µm. Data are representative of at least three independent experiments and represent mean ± SD. (ns, no statistical significance; **P<0.01; ***p < 0.001).