Fig. 5: siRNA depletion of the H2A.X poly(A) mRNA. | Nature Communications

Fig. 5: siRNA depletion of the H2A.X poly(A) mRNA.

From: Dual RNA 3’-end processing of H2A.X messenger RNA maintains DNA damage repair throughout the cell cycle

Fig. 5

a Schematic of siRNA binding sites for 3’-UTR extension of H2A.X poly(A) mRNA. siRNA: single siRNA. siRNAp: pool of four different siRNAs. b H2A.X mRNA northern blot of total RNA and quantification from HeLa cells treated with indicated siRNAs for 72 hr. Mean ± SD presented for n = 3 independent experiments, two-way ANOVA with Tukey’s multiple comparisons test. ***P-value ≤ 0.001, ****P-value ≤ 0.0001. H4 SL: core H4 histone SL mRNA. 18S rRNA used as loading control (left). Total protein extract western blot and quantification. Actin and core histone H4 are controls. H2A.X indicates total H2A.X protein levels. The two 15 kDa marks indicate where the membrane was cut. Mean ± SD presented for n = 3 independent experiments, paired Student’s t-test, **P-value ≤ 0.01 (top right). Percentages of cells in G1, S and G2/M analysed by flow cytometry in siRNA-treated HeLa cells. Staining for flow cytometry was performed using propidium iodide. Mean ± SD presented for n = 3 independent experiments, two-way ANOVA with Dunnett’s multiple comparisons test, ns: not significant P-value >0.05, ****P-value ≤ 0.0001 (bottom right). c H2A.X mRNA northern blot of total RNA from RPE-1 cells depleted of H2A.X poly(A) mRNA and synchronised by contact inhibition (time following block release). H4 SL: core H4 histone SL mRNA. EEF1A1 mRNA and 18S rRNA used as loading controls. d Percentage of S-phase cells in RPE-1 cell population treated with either siLuc (dark grey bars) or siRNAp (light grey bars) and synchronised by contact inhibition. X-axis represents time points in hours after release from cell cycle arrest and y-axis percentage of S-phase cells determined by EdU labelling followed by flow cytometry (Supplementary Fig. 5). Mean ± SD presented for n = 4 independent experiments. Unpaired Student’s t-test ns: not significant P-value > 0.05, **P-value ≤ 0.01, ***P-value ≤ 0.001. e H2A.X mRNA northern blot of total RNA from RPE-1 cells treated with indicated siRNAs and arrested for 4 days and harvested at indicated time points after resuming cell cycle. 5 μM etoposide added at 6 hr (G1) for a 2 hr period. H4 SL: core H4 histone SL mRNA. EEF1A1 mRNA and 18S rRNA used as loading controls. Same samples were analysed by PFGE. Dark blue box represents ethidium bromide-stained PFGE agarose gel. Light blue regions correspond to saturated DNA signal in gel well.

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