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. Author manuscript; available in PMC: 2018 Jan 23.
Published in final edited form as: Curr Biol. 2016 Dec 15;27(2):210–223. doi: 10.1016/j.cub.2016.11.049

Figure 3. Partial depletion of multiple repair factors leads to DNA damage accumulation and chromosomal variation.

Figure 3

(A) Key nuclear or DNA repair transcripts from RNA-seq analysis of U2OS cultures, either pre- or post-migration. Mouse knockouts/mutations of key genes cause copy number variations. MYH6 (a cardiac specific gene) shows zero reads. (Normalization to Reads per kilobase million, n=8 samples).

(B) Simultaneous partial knockdown of four repair factors (si4, red) versus control (siCtrl, gray) when normalized to non-treated (NT). 25 nM of each siRNA (red) was used except for siRPA1 titration (Figure S2). Protein levels quantified by immunoblots were normalized by housekeepers (HSP90, beta-actin), with BRCA1 quantified by immunofluorescence. (n=3 blots or ≥150 nuclei, n=3 expts, *p<0.05).

(C) DNA damage increased in 2D cultures of si4 cells based on γH2AX foci (i, Figure S2) and comet assay (ii). siCtrl slightly increases damage, perhaps because of lipofectamine. DNA damage sites induced by 1 hour 10μM etoposide treatment are resolved by the siCtrl cells within hours, but γH2AX foci count of si4 cells remained at a high number (iii, ≥110 nuclei per condition, n≥3 expts, *p<0.05 vs. NT, #p<0.001 vs siCtrl).

(D) si4 increases γH2AX foci regardless of 3-μm pores migration, while damage increases in controls (i, ≥ 50 nuclei per condition, n≥3 expts, *p<0.05 between top and bottom). Migrated %-ages are similar for all conditions (ii, Figure S2; ≥3 transwell per condition, n≥3 expts).

(E, F) DNA content from Hoechst-33342 intensity shows 2N and minor 4N peaks for controls but shifts for si4 indicate poly- or aneu-ploidy (Figure S2, ≥160 nuclei per condition, n≥3 expts; *p<0.001 in two-sample Kolmogorov-Smirnov (KS) test for higher modes, with α=0.05 [50]). Metaphase spreads show higher ploidy of U2OS cells versus diploid MSCs, and more chromosome segments for si4 cells (i). Both Chr-1 length and segment counts of U2OS are higher versus MSCs, but (ii) Chr-1 counts are unaffected by si4 (≥20 spreads per condition, n≥3 expts, *p<0.05 MSC versus U2OS, #p<0.05 U2OS versus U2OS).