Fig. 4

Derivation and characterization of HGPS-MSCs. a Immunostaining of SSEA-4, TRA60-1 and NANOG and OCT3/4 (Scar bar 10 μm), alkaline Phosphatase (AP) activity and embryoid body (EB) formation of HGPS-iPSCs. Scar bar 100 μm. b DNA methylation in the genomic region at OCT3/4 promoter in HGPS-iPSCs and its parental dermal fibroblasts. Open and filled circles represent unmethylated and methylated CpGs, respectively. Each row represents bisulfite sequencing result from a given amplicon and ten amplicons were analyzed. c Immunostaining of lamin B1 in HGPS-iPSCs and WT-iPSCs. Scar bar 10 μm. d Expression of nuclear lamins and HDAC1 examined by Western blotting. β-Actin serves as a loading control. e Co-immunostaining of lamin B2 and progerin in HGPS-iPSCs derived MSCs, scar bar 10 μm. Percentages of cells exhibiting nuclear deformation and progerin expression were analyzed. Data represent mean ± S.E.; n = 4; *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.001*p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; ns, not significant; unpaired two-tailed Student’s t-test is used for statistical analyses. f Immunoblotting of Ki 67 in HGPS-iPSCs and WT-iPSCs derived MSCs. Scar bar 100 μm. Data represent mean ± S.E.; n = 5; *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.001; ns, not significant; unpaired two-tailed Student’s t-test is used for statistical analyses. g SA-β-gal staining of MSCs derived from HGPS-iPSCs and WT-iPSCs. Cellular senescence was quantified by SA-β-gal positive cell number. Data represent mean ± S.E.; n = 5, *p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns, not significant; one-way ANOVA coupled with Tukey’s post hoc test is used for statistical analyses. h Transcription of p16, p21 and SASP-related inflammatory cytokine examined by qPCR. Data represent mean ± S.D.; n = 4; *p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns, not significant; one-way ANOVA coupled with Tukey’s post hoc test is employed for statistical analyses