Offspring of rnaseh2a−/− adults have severe developmental defects, increased DNA damage, increased ribonucleotides and an upregulated inflammatory response. (A) Acridine Orange staining of apoptotic cells (green) in 24hpf embryos from homozygous and wild-type incrosses. (B) Quantification of acridine orange positive cells from (A). Unpaired t-test, ****P <0.0001, ±SD (n = 12, single embryo). (C) Schematic of single ribonucleotide cleavage assay. (D) Whole embryo lysate from 24hpf embryos were incubated with dsDNA substrate containing a single ribonucleotide. Positive and negative controls were purified RNaseH2 and lysis buffer respectively. DNA was tagged with Cy5. (E) Quantification of cleavage (%) in activity assay, ±SD, one-way ANOVA, P <0.05 (n = 3, 20 pooled embryos). (F) Gel of alkaline (NaOH) and control (NaCl) treated DNA of 24hpf embryos from homozygous and wild-type crosses. (G) Densitometry plot of DNA intensity produced with R. (H) Image quantification showing ratio of long and short fragments. Student's t-test **P< 0.01, ± SEM, n = 3. (I) γH2AX staining (green) in tails of 24hpf rnaseh2a−/− embryos. Nuclei stained with DAPI (blue). (J) Quantification of γH2AX foci per nucleus in tail tip of individual embryos (>100 nuclei per embryo, 3 or 4 per genotype shown). Red lines show average per embryo and SEM. Unpaired t-test, Welch's correction, ***P <0.001, n ≥ 3). (K) qPCR of interferon stimulated genes (ISG15, mxa, IFNΦ), proinflammatory response markers (TNFa, ILb, IL6) and senescence markers (p21). ****P <0.0001, ***P <0.001, **P <0.01, ±SD (n = 3, 20 pooled embryos).
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