Figure 2

Ablation of SIRT6 in hMSCs results in elevated ROS levels and increased vulnerability to oxidative injury. (A) WT and SIRT6-deficient hMSCs were treated with vehicle (DMSO) or 50 μM PX-12 for 24 h, and the apoptotic cells were determined by Annexin V-PI staining via FACS. (B) Statistical analysis of A. Apoptotic cell percentage in vehicle-treated WT hMSCs was normalized to 1. Data were presented as mean ± SEM, n = 3, NS, not significant, *P < 0.05. (C, D) Cellular reactive oxygen species (ROS) and 8-oxodG levels were determined by staining with H2DCFDA probe (C) and an anti-8-oxodG antibody (D), respectively, and measured by FACS. (E) SIRT6-deficient hMSCs were pretreated with H₂O (control) or 1 mM NAC for 1 week, and then were treated with vehicle (DMSO) or 50 μM PX-12 for 24 h. Cellular apoptosis was measured by Annexin V-PI staining. Data were presented as mean ± SEM, n = 3, **P < 0.01. (F) Overexpression of WT SIRT6 (SIRT6 (WT)), not SIRT6 H133Y mutant (SIRT6 (HY)), in SIRT6-deficient hMSCs partially restored cellular ROS to normal levels. A luciferase (Control)-expressed vector was used as control. (G, H) SIRT6-deficient hMSCs were transduced with SIRT6 (WT), SIRT6 (HY), or Control vector, and then cells were treated with vehicle (DMSO) or 50 μM PX-12 for 24 h. Cell viability (G) and cellular apoptosis (H) were measured by MTS assay and Annexin V-PI staining, respectively. Data in G and H were presented as mean ± SEM, n = 6, *P < 0.05, **P < 0.01, ***P < 0.001.