Figure 2.

Suppression of H3K9 Demethylases KDM3A and KDM4C Induces DNA Damage and Accelerates Cellular Senescence in hUCMSCs

Two different hUCMSCs lines (hUC009, hUC013) were used for knockdown or IOX experiments.

(A) Representative images of colony formation and β-Gal staining (scale bar = 100μm) in hUCMSCs treated with control shRNA or shRNAs targeting KDM3A or KDM4C. The cells were selected by ZsGreen positivity after lentiviral transduction and grew for two more passages. Quantification of β-Gal staining is shown at the right; data are presented as the mean ± SEM. ***p < 0.001 (t test: n = 3).

(B) RT-qPCR assay showing the expression levels of KDM3A, KDM4C, p15, and p21 after shRNA treatment; data are presented as the mean ± SEM. **p < 0.01; ***p < 0.001 (t test: n = 3).

(C) Representative images (scale bar = 5μm) and quantification of 53BP1, γ-H2AX foci in hUCMSCs transduced with lentiviral particles carrying shKDM3A-1, shKDM4C-1, or control shRNA. Quantification data are presented as mean ± SEM of values from three independent experiments with triplicate wells analyzed on 6–8 cells/field from five different fields. ***p < 0.001 (t test).

(D) MTT assay of hUCMSCs (p6) treated with different concentrations of IOX1 or DMSO for 72 h. Data are presented as the mean ± SEM of values from three independent experiments; **p < 0.01, IOX70μM compared with control; ##p < 0.01, IOX100μM compared to control group (t test).

(E) Quantification of β-Gal staining in hUCMSCs (p7) treated with different concentrations of IOX1 (20, 50, 70, and 100 μM). Data are presented as the mean ± SEM. *p < 0.05; **p < 0.01 (one-way ANOVA, n = 3).

(F) Quantification of 53BP1 and γ-H2AX foci in hUCMSCs (p6) treated with different concentration IOX1 (20, 50, 70, and 100 μM) and DMSO control. Quantification is shown at the right; mean ± SEM of values from three independent experiments with triplicate wells analyzed on 6–8 cells/field from five different fields. *p < 0.05; **p < 0.01 (one-way ANOVA).