Figure 4.

Doxorubicin Induces Heterochromatin Reorganization and DNA Damage Response

Two hUCMSCs lines (hUC009, hUC013) were used for Doxorubicin-induced cellular senescence model.

(A) Early passage hUC-MSCs (p6-7) were treated with Doxorubicin for 48 h. Representative Western blot showing the expression levels of KDM3A, KDM4C, heterochromatin marks, NCAPD2, NCAPG2, and senescence marks at different time points (0–48 h) after Doxorubicin treatment.

(B) Representative immunofluorescence images of 53BP1 or γ-H2AX foci and H3K9me2/3 staining (scale bar = 5μm) in hUCMSCs treated with Doxorubicin for different time points.

(C) Quantification of 53BP1 or γ-H2AX and H3K9me2/3 immunofluorescence staining in hUCMSCs treated with Doxorubicin for different time points (CTCF, corrected total cell fluorescence). Data are presented as mean ± SEM of values from three different experiments with triplicate wells analyzed on 6–8 cells/field from five different fields; *p < 0.05; **p < 0.01, 53BP1, γ-H2AX compared with control group, #p < 0.05; ##p < 0.01, H3K9me2/3 compared with control group (Wilcoxon/Mann-Whitney test).

(D) Representative images and quantification of 53BP1 and γ-H2AX immunofluorescence staining (scale bar = 5μm) in Doxorubicin-treated hUCMSCs transfected with scrambled siRNAs or siRNA mixtures (siKDM3A-1 + siKDM4C-3). Data are presented as mean ± SEM of values from three different experiments with triplicate wells analyzed on 6–8 cells/field from five different fields; **p < 0.01; ***p < 0.001 (t test).

(E) Representative images and quantification of β-Gal staining (scale bar = 100μm) in control group or Doxorubicin-treated hUCMSCs transfected with Vector plasmid or KDM3A, KDM4C, or NCAPD2 plasmid.

Data are presented as the mean ± SEM. *p < 0.05; **p < 0.01 (t test, n = 3).