Fig. 6: Loss of SFPQ mediates defects in mitotic progression.

ChIP on U-2 OS cells transfected with control and SFPQ-specific siRNAs. The graphs report the binding of ATR (A) and γH2AX (B) to the indicated region of interest. Representative images of native metaphase spreads obtained from control and SFPQ knock-down U-2 OS silenced with indicated siRNAs. Immunostaining was carried out using anti p-ATR(Thr1989) and anti-CREST (centromere) antibodies (C) or anti-p-RPA32(Ser33) and anti-CREST antibodies (D). Left panels, representative images. Arrowheads indicate position of p-RPA32Ser33/p-ATR staining in vicinity to centromeres. Right panels, quantification of positive staining at (peri)-centromeric regions. E Immuno-RNA FISH staining of native metaphase chromosomes derived on siRNA-transfected U-2 OS cells. Staining with anti-CREST antibody and a pericentric SatIID RNA-FISH probe is shown. Left panel, representative images. Right panel, quantification of SatIID signals at (peri)centric regions; Arrowheads, position of SatIID RNA-FISH staining in vicinity to centromeres. F Representative images of time-lapse microscopy using U-2 OS cells constitutively expressing GFP-tagged histone H2B after siRNA transfection. Left panels, representative images of observed mitotic defects. Right panels, relative quantifications of mitotic defects. G Giemsa-stained metaphases of U-2 OS cells transfected with indicated siRNAs to evaluate rates of sister chromatid exchange (SCE) and chromosome breaks. Left panels, representative images. Right panels, quantification of metaphase chromosome defects. Zoomed images, observed defects. Bars in data blots represent mean values, error bars indicate standard deviation. N = number of independent experiments. An unpaired, two-sided Student’s t test was used to calculate statistical significance; p values are shown. For (C–E), n= number of analyzed metaphase spreads, n# = number of analyzed chromosomes, graph shows median and 10–90 percentile; dots, outliers. Scale bars correspond to (C–E), 0.1 μm; (F) 1 μm and (G) 5 μm. Scale bars apply to all images in the respective immunofluorescence panels. C–E DNA (blue) was stained using DAPI (4’,6-diamidino-2-phenylindole). For (F), n= number of analyzed nucleus (used for micronuclei quantification), n# = number of analyzed cell divisions (used for chromatin bridges and multilobed cells qualifications). For G, n = number of analyzed metaphases. Source data are provided with this paper.