Fig. 6: Effects of H2A.X poly(A) mRNA depletion under DNA damage conditions.

a H2A.X mRNA northern blot of total RNA from HeLa cells treated with siRNA for 72 hr (left panel). H4 SL: core H4 histone SL mRNA. EEF1A1 mRNA, 28S and 18S rRNA used as loading controls. Etoposide treatment (5 μM) was performed for 2 hr before harvesting. Tubulin was used as loading control. H2A.X: total H2A.X protein. γH2A.X: H2A.X phosphorylated at Ser139. M: protein size marker. H2A.X poly(A) mRNA quantification in siLuc without and with etoposide by real-time PCR (right panel). Mean ± SD presented for n = 3 independent experiments, paired Student’s t-test, ns: not significant P-value >0.05. b Western blot of total protein extracts from HeLa cells as indicated. Etoposide treatment (5 μM) was performed for 2 hr before harvesting. Black arrows: total H2A.X protein in siLuc control cells before and after etoposide treatment. c IF of HeLa cells with etoposide treatment (5 μM for 2 hr before harvesting). Blue: DAPI (nuclei). Red: γH2A.X. White: EdU (S-phase cells). Quantification of nuclear γH2A.X signal in non-S-phase (black bars) and S-phase cells (grey bars) (bottom). Mean ± SD presented for n = 3 independent experiments, two-way ANOVA with Tukey’s multiple comparisons test. ****P-value ≤ 0.0001 (left). d–f As in a–c, respectively, but for HCT-116 cells. The red asterisk indicates unequal loading in lane 1 of d.