Identification of H2B S6ph phosphatases. (A) RPE-1 cells were arrested for 16 h with RO-3306 and then released for 1 h in the presence of nocodazole and okadaic acid (1 µM), LB-100 (10 µM), or DMSO (control) for 1 h. Chromosome spreads were analyzed by immunofluorescence for H2B S6ph and CENP-A localization. Bars: 1 µm (magnification); 5 µm (main). (B) Cells were transfected with siRNAs targeting catalytic subunits of PP1α, β, and γ or control siRNAs (Origene). Knockdown efficiency was confirmed by qPRC (see Fig. S3 B). After 24 h, cells were arrested at G1/S by a thymidine block and released, followed by the addition of nocodazole for 6.5 h. The mitotic cells were further treated for 30 min with AZD1152 or the DMSO control and stained as shown. Bars, 2 µm. (C) The experiment was performed as in B, and fluorescence intensities (given in artitrary units; A.U.) were quantified from 10 representative cells per condition using ImageJ and GraphPad Prism. (D) The recombinant H2B protein was phosphorylated by an in vitro kinase assay as described for Fig. 6 A. CDK1-cyclin B1 was removed from the extracts by immuno-depletion and the supernatant containing 2 µg of phosphorylated H2B protein was taken and incubated with 0.2 µg of recombinant PP1α (Novus Biologicals) or GST control (Sigma) for 30 min at 37°C. The reaction was analyzed for histone phosphorylation by Western blotting (top) and protein integrity by SDS-PAGE and CBB staining as shown.
