Extended Data Fig. 1: ADAR1-mediated A-to-I RNA editing is primarily observed during S phase and is regulated by ROS.

(a) U2OS cells were treated with different doses of NAC for 24 h. 10 μM EdU was added for 10 min prior to immunofluorescence staining with anti-γH2AX and anti-biotin antibodies. γH2AX levels were quantified in EdU-positive cells. Statistical analysis of the foci number was performed using one-way ANOVA tests. Data are mean ± SDs. 60 events quantified in each condition are shown. n = 3 biologically independent experiments. Scale bars, 10 μm. (b) U2OS cells were treated with different doses of H2O2 for 30 min. 10 μM EdU was added for 10 min prior to immunofluorescence staining with anti-γH2AX and anti-biotin antibodies. γH2AX levels were quantified in EdU-positive cells. Statistical analysis of the foci number was performed using one-way ANOVA tests. Data are mean ± SDs. 60 events quantified in each condition are shown. n = 3 biologically independent experiments. Scale bars, 10 μm. (c) Immunofluorescence analysis of ADAR1 p110 and p150 in MDA-MB-231 cells. Scale bars, 20 μm. (d) Left: MDA-MB-231 cells were pulse-labeled with 10 μM EdU for 15 min, and then incubated with thymidine (dThd) for 0 or 30 min as indicated, followed by treatment with or without 5 mM HU for 4 h. iPOND samples were collected and analyzed by western blot. Right: quantification of mean EdU intensity from immunofluorescence assays under the same conditions. Data are the means ± SDs. Statistical analysis was performed using one-way ANOVA tests. n = 3 biologically independent experiments, with 1,000 cells quantified per experiment. (e) Sanger sequencing results of the ADAR1 locus in MDA-MB-231 cells with ADAR1 WT or ADAR1 KO. (f) ADAR1 WT and KO MDA-MB-231 cells were treated with 10 μM EdU for 10 min, followed by treatment with either DMSO or 5 mM HU for 4 h, and then subjected to PLA using anti-ADAR1 and anti-biotin antibodies. Representative images of PLA foci are shown. Data are mean ± SDs. Statistical analysis was performed using two-tailed unpaired t-tests. 60 cells quantified in each group were obtained from one experiment. n = 3 biologically independent experiments. Scale bars, 10 μm. (g) Using the RNAG editing reporter system, GFP expression is suppressed by a premature stop codon unless adenosine is edited to inosine. MDA-MB-231 cells stably expressing the RNAG reporter were incubated with EdU (10 μM) for 10 min to label S-phase cells. GFP expression, indicating RNA editing activity, was detected by immunofluorescence. Scale bars, 100 μm. Data are the means ± SDs. Statistical analysis was performed using two-tailed unpaired t-tests. n = 3 biologically independent experiments, with 1,000 cells quantified per experiment. (h) Cell cycle progression assay in MDA-MB-231 cells bearing the RNAG reporter. Top, experimental schema to test cell cycle progression. Bottom, expression level of Cyclin A2 and Cyclin D1 was checked by western blot at indicated time points after release from G2/M block. (i, j) ADAR1 WT and KO MDA-MB-231 cells bearing the RNAG reporter were subjected to IF at the indicated time points after G2/M release to evaluate RNA editing levels (i). Quantification of RNA editing levels at different cell cycle stages is shown (j). Data are mean ± SDs. n = 3 biologically independent experiments. Scale bars, 5 μm. (k) Immunoblot analysis of ADAR1 in the indicated MDA-MB-231 cells, which were supplemented with the specified construct. (l) Quantification of RNA editing levels in the indicated MDA-MB-231 cell lines treated with or without 10 mM NAC for 24 h. Data are the means ± SDs. Statistical analysis was performed using two-tailed unpaired t-tests. n = 3 biologically independent experiments, with 1,000 cells quantified per experiment.

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