Figure 3.

Spliceosome depletion results in increased DNA damage and checkpoint-dependent G2 arrest. (A) Analysis of DNA damage by immunostaining. HeLa cells were transfected with the indicated siRNAs. Cells were fixed at 48 h after transfection and stained with antibodies against γ-H2AX and DAPI. Sample micrographs are shown. (B) Quantification of the percentage of γ-H2AX-positive cells in (A). (C) Quantification of nuclear γ-H2AX intensity in (A). Each dot represents one nucleus. (D) HeLa cells were transfected with Control (siControl) or siSNRPB-3 siRNA together with siRNAs against the indicated DNA damage checkpoint components. Western blots of total lysates blotted with the indicated antibodies are shown. (E) Quantification of mitotic entry. HeLa cells transfected with the indicated combinations of siRNAs were incubated with taxol at 24 h after siRNA transfection. Samples were collected after 15 h in taxol, and the cell cycle profile was analyzed by flow cytometry using Propidium Iodide (PI) and anti-MPM2 staining. The percentage of mitotic cells (MPM2+) is indicated.