Figure 5.

Inhibition autophagy results in stemness injury and ROS increase in irradiated MSCs. (a) LC3 expression was detected by western blotting assay in irradiated MSCs pretreated with starvation by autophagy inhibitor 3-MA or CQ. (b) Irradiated MSCs pretreated with starvation were transfected with shRNAs to knockdown the autophagy-associated genes ATG7 and Beclin1. The bottom panel is a GAPDH-loading control. (c) CFU-F assays. The number of colonies was determined after 14 days of culture. (d) The expression of stemness markers Nanog, Oct4 and Sox2 of irradiated MSCs pretreated with starvation by inhibiting autophagy were measured by real-time PCR and western blotting. (e) Osteogenic differentiation of irradiated MSCs pretreated with starvation by autophagy inhibitors was stained with Alizarin Red S. (f) The expression of osteogenic markers ALPL, OGN and RUNX2 were measured by real-time PCR. (g) Adipogenic differentiation of irradiated MSCs pretreated with starvation by autophagy inhibitors were stained with Oil red-O. (h) The expression of adipogenic markers LPL, CFD and PPAR-γ were measured by real-time PCR. (i) Irradiated MSCs pretreated with starvation by autophagy inhibitors stained with DCF-DA to detect ROS level were measured by immunofluorescence. Cell nucleus was stained with Hoechst 33258. (j) Irradiated MSCs pretreated with starvation by autophagy inhibitors were stained with γ-H2A.X antibody to determine DNA damage. Cell nucleus was stained with DAPI. The data presented are from three replicates as mean±S.D. *P<0.05