Fig. 6: The phospho-dead RPA^SA/SA mutant exhibits reduced phosphorylation of Histone H3 and Aurora B.

a Western blot represents a decrease in Ser10 Histone H3 phosphorylation in RPA^SA/SA mutant relative to wild type cells. Asynchronous (async) cells were assayed as control. Blots are representative of three independent experiments. b Blots represented in (a) were quantitated and normalized to loading control (Tubulin). Data are expressed as a fold-over RPA^WT/WT. Data are presented as mean of three independent experiments and error = SEM. Statistical significance was determined using an unpaired two-tailed t-test: **p = 0.0016. c Western blot represents total Histone H3 levels in RPA^SA/SA mutant and parental cells. Blots are representative of three independent experiments. RPA^WT/WT (d) cells synchronized in mitosis and collected by shake-off method were found to be arrested in G2/M phase of cell cycle similar to RPA^SA/SA mutant (e) as analyzed by flow cytometry. DNA content was determined using propidium iodide staining. Data represent three independent experiments. f Surface area of nuclei stained for phospho Ser-10 Histone H3 represented in supplementary Fig. 7b. were quantitated using Image J analysis. Data are presented as a scatter plot of more than 200 nuclei measured across three independent measurements. Error = SEM. Statistical significance was determined using an unpaired two-tailed t-test. p = 0.00000000001. g Blots represent the changes in Aurora B kinase activity as determined by T232 autophosphorylation and total levels. Vinculin = loading control. h Plot depicts decreased Aurora B activity in the RPA^SA/SA mutant relative to WT in mitotic cells. Asynchronous cells were used as controls. Aurora B-T232 phosphorylation levels were normalized to the loading control. Data are representative of three experiments. Error=SEM. Statistical significance was determined using an unpaired two-tailed t-test: ****p = 0.000087. i Schematic shows the feedback regulation of Aurora B activity through Ser-384 RPA70 phosphorylation in mitosis. However, the precise mechanism of regulation remains to be determined.