Fig 1: PP4 and CNTRB act together during DNA damage response. (a) Enumeration of γH2AX foci in HeLa cells. Cells were transfected with the following siRNAs: siControl, siCNTRB, siPP4c, siR3A, siR3B, or co-transfected with siCNTRB and siPP4c. After 48 h of RNAi, DNA damage was induced with 2 Gy X-ray irradiation and γH2AX foci were counted in 50 cells at different times (2, 4, and 8 h) post-irradiation. NT indicates the non-treated (non-irradiated) samples. Graphs include all data points and mean ± SEM (n = 3, three replicates). Asterisks indicate p-values (*** p < 0.001) obtained by linear regression fitted independently for each time point. (b) Homologous recombination measuring assay. Cells were transfected with the following siRNAs: siControl, siCNTRB, siPP4c, siR3A, siR3B, or co-transfected with siCNTRB and siPP4c. After 48 h, the cells were transfected with I-SceI expressing plasmid construct. The number of GFP-positive cells was calculated with Cellprofiler [52] and normalized to siControl-transfected cells. Graphs include all data points and mean ± SEM (n = 3, three replicates). Asterisks indicate p-values (*** p < 0.001) obtained by linear regression fitted independently for each time point. (c,d) Chromosome study in CNTRB- or PP4 subunit-depleted cells. Cells were transfected with the following siRNAs: siControl, siCNTRB, siPP4c, siR3A, siR3B, or co-transfected with siCNTRB and siPP4c. After 48 h, the cells were treated with 2 Gy X-ray irradiation and treated for 6 h with 20 µg/mL caffeine and 10 µg/mL colchicine. NT refers to non-treated (non-irradiated) cells. (c) Chromosome aberrations were counted in 150 spreads in each sample, and the percentage of arms closed phenotype was plotted. Graphs include all data points and mean ± SEM (n = 3, three replicates). Asterisks indicate p-values (*** p < 0.001) obtained by linear regression fitted independently for each time point. (d) Representative images of chromosome spreads isolated from the indicated cells in NT or in X-ray irradiated samples. Scale bar is 10 µm. Insets at bottom left show magnified representative chromosome morphologies.