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. 2021 Mar 12;12:1654. doi: 10.1038/s41467-021-21921-x

Fig. 1. ADAR1 depletion resulted in abnormalities of the nucleus and upregulation of DNA damage pathway and cell cycle marker genes.

Fig. 1

a HeLa cells were first transfected with siControl or siADAR1 (siADAR1-1) for 72 h and then treated with CellLight Tubulin-GFP. Nuclei were visualized by staining of DNA with SiR-DNA reagent. Representative images taken from real-time videos (Supplementary Movies S1 and 2) are presented. Scale bar, 50 μm. b The frequency of abnormalities of the nucleus (nucleoplasmic bridge, micronuclei, and multinuclei) was estimated by examining at least 200 individual HeLa cells treated with siControl or siADAR1-1 RNAs. Values are mean ± standard error (n = 3, biologically independent samples) with significant differences by two-tailed Student’s t test indicated, **P < 0.01. Scale bar, 5 μm. c Telomere DNA damages in ADAR1-depleted cells. Telomere FISH and immunostaining for γH2AX revealed significantly increased telomere dysfunction-induced foci (TIF, indicated by yellow arrowheads), suggesting the causative relevance of the telomeric repeat DNA damage to chromosome abnormality detected in ADAR1-depleted HeLa cells. At least 200 individual HeLa cells treated with siControl or siADAR1-1 RNAs were examined. HeLa cells with one or more TIFs were counted as TIF-positive cells. Values are mean ± SD (n = 3, biologically independent samples) with significant differences by two-tailed Student’s t test indicated, **P < 0.01. Scale bar, 10 μm. b, c All individual experimental data values and exact P values are presented in Source Data file. d Western blotting analysis was done using total cell extracts from HeLa cells treated with siControl or two separate siADAR1 (siADAR1-1 and -2) RNAs for 72 h. Protein molecular weight markers are presented in Source Data file. e The R-loop structure consisting of an RNA:DNA hybrid formed between the RNA strand newly transcribed by RNA polymerase II and the template DNA strand with the single-stranded antisense DNA bound with ssDNA-binding protein RPA as well as major regulators are schematically shown.