Research Paper Volume 12, Issue 7 pp 5693—5715

Indian Hedgehog regulates senescence in bone marrow-derived mesenchymal stem cell through modulation of ROS/mTOR/4EBP1, p70S6K1/2 pathway

Figure 8. mTOR and ROS inhibition reversed IHH depletion-induced senescence-related signaling pathways, cell cycle arrest, and inhibited CFU. siRNA IHH-transfected BMSC (n=5) were incubated with or without INK128, DPI, or NAC for 48hours. P53 (A), P16 (B), PI3K, p-PI3K (C), Akt1, p-Akt1 (D), NF-κB, p-NF-κB (E), STAT3, and p-STAT3 (F) proteins expressions were measured by Western Blot. β-actin was used as an internal control. (G) BMSC (n = 5) were treated with or without INK128, DPI, or NAC in presence of siRNA IHH for 24hours. Fixed cells stained by PI and RNase A, and then analyzed by flow cytometry for cell cycle distribution. (H) BMSC (n = 5) were treated with or without INK128, DPI, or NAC in presence of siRNA IHH for 24hours and then incubated in 10% FBS in MEM-ALPHA medium for 12 days. Colonies were visualized after staining with 0.02% crystal violet stain. All results were normally distributed and shown as mean ± SEM. *P <0.05, **p <0.01, ***p <0.001, ****p <0.001.