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. 2023 Jun 15;30(7):1811–1828. doi: 10.1038/s41418-023-01182-5

Fig. 5. Knockdown of HDAC3 results in R-loop accumulation that triggers DNA damage.

Fig. 5

A and B Western blot analysis of co-IP between Myc-NKAP and Flag-HDAC3 proteins expressed in HEK293T cells. The IP was performed using anti-Myc (A) or anti-Flag (B) antibodies with cell lysates treated with (+) or without (−) RNase A. C Western blot analysis of co-IP between endogenous HDAC3 and NKAP proteins in U2OS cells. The IP was performed using anti-HDAC3 antibody with cell lysates. D–F Representative images (D) and quantification (E and F) of S9.6 fluorescence staining in cells following transfection with control or HDAC3 siRNAs for 48 h. anti-Nucleolin antibody was used to detect Nucleolin and label the nucleolus. The graph shows the S9.6 fluorescence intensity per nucleus (E) and nucleoplasm (F). The value for the nucleoplasm was determined by the signal intensity per nucleus with subtraction of the nucleolar signal. ****P < 0.0001 (Mann–Whitney U-test, two-tailed). G Dot blot analysis of R-loops using the S9.6 antibody. Genomic DNA was extracted from cells following transfection with control or HDAC3 siRNAs for 48 h. RNase H treatment was conducted alongside and used as a negative control. dsDNA was used as an internal sample loading control. H DRIP-qPCR analysis of the indicated loci in cells following transfection with control or HDAC3 siRNAs for 48 h. In vitro RNase H treatment was conducted prior to pulldown and served as a control. The graph shows the relative value of R-loop level in each gene loci which was normalized with respect control. ****P < 0.0001; **P < 0.01; *P < 0.05 (paired Student’s t test). I and J Representative images (I) and quantification (J) of γH2AX foci in cells following transfection with control or HDAC3 siRNAs for 48 h and subsequent treatment with (+) or without (−) doxycycline (DOX) to induce RNH1 overexpression. The graph shows the number of foci per cell. ****P < 0.0001; ***P < 0.001 (Mann–Whitney U-test, two-tailed). K Western blot analysis using indicated antibodies. Lysates were prepared from cells following transfection with control or HDAC3 siRNAs for 48 h and subsequent treatment with (+) or without (−) doxycycline (DOX) to induce RNH1 overexpression. GAPDH serves as a loading control. Scale bar, 20 μm. a.u. indicates arbitrary units. Data are presented as Mean ± standard error of the mean (SEM).