Fig. 6
From: Deacetylation of FOXP1 by HDAC7 potentiates self-renewal of mesenchymal stem cells
hFOXP1T176G mutation potentiates the replicative capacity of hMSCs. a A murine FOXP1-T172G substitution mutant transfected into 293 T cells is enriched in global acetylation relative to FOXP1 wild type (WT) control. Proteins were pulled down via their His-tags by anti-His, fractionated on SDS-PAGE and then blotted with either pan anti-Acetyl-Lysine antibody (Ace) or for loading control antibodies specific for HA tag or for β-actin. b The ubiquitination levels of FOXP1-T172G were increased as compared to FOXP1 controls following transfection and fractionation in 293 T cells as described in panel A. c Schematic diagram depicting the strategy employed for hFOXP1T176G engineering in human Embryonic Stem Cells (hESCs) via transfection with episomal Cas9n/sgRNA and donor vectors. d Validation of hFOXP1T176G-engineered hESCs by Sanger sequencing. The mutated leucine to glycine codon (GGC) is boxed in red (lower panel) relative to the wild type codon (ACC) shown in the left panel as black. e Crystal purple staining identifies CFU-F-positive colonies of hMSCs at passages P3 and P10. hMSCs shown were directly induced from hFOXP1T176G-engineered hESCs. Bar, 100 μm. f Population doubling curve of hFOXP1T176G-engineered hMSCs. Results shown are representative of 3 independent measurements. g Q-PCR analysis of cellular senescence as indicated by levels of cell cycle (p16, p21, p27) and tumor repressor (p53, Rb) transcript levels within hMSCs at P15. h SA-β-gal staining identifies cellular senescence of hFOXP1T176G-engineered hMSCs at P10 and P15. Data are representative of 3 independent measurements. Bar, 100 μm. i DNA damage as measured in hMSCs at P15 by immunofluorescence of Lamina-associated polypeptide 2 (LAP2) (left panel). Right panel, quantification of γH2AX-positive cells. n = 3. Bar, 100 μm. j Immunofluorescence of γH2AX in hMSCs at P15. Right panel is quantification of γH2AX-positive cells. Results shown are representative of 3 independent measurements. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ns, non-significant. Bar, 100 μm. k Schematic diagram showing that deacetylation of acetylation marks (blue dots) from FOXP1 by HDAC7 potentiates its effect on promoting self-renewal (indicated by the red arrow) of MSCs