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 9. Biased differentiation induced by IHH shortage was corrected after mTOR and ROS inhibition in BMSC. (A) Morphology of siRNA IHH BMSC with and without INK128, DPI, or NAC. (Ba, b), (D, E) siRNA IHH-transfected BMSC (n=5) were incubated with or without INK128, DPI, or NAC for 24hours and then incubated in adipogenesis differentiation medium for 21 days. Adipocytes were visualized under inverted light microscope and adipogenesis measured by staining fat droplets using Oil-Red-O stain. (C and F) siRNA IHH-transfected BMSC (n=5) were incubated with or without INK128, DPI, or NAC for 24hours and then incubated in osteogenic differentiation medium for 21 days. Osteogenesis measured using Alizarine-Red-S stain. (G) RT-PCR for adipogenesis markers, PPARγ, LPL, and FABP4, and osteogenesis markers, ALP-BMA, RUNX2, and osteocalcin in IHH-depleted BMSC after incubation with or without INK128, DPI, or NAC for 24hours. GAPDH was used as a housekeeping gene. All results presented as mean ± SEM. *P <0.05, **P < 0.01, ***p <0.001, ****p <0.0001.