Extended Data Fig. 9: Depletion of RECQ1 prevents accumulation of cytosolic ssDNA in SAMHD1-depleted cells.
From: SAMHD1 acts at stalled replication forks to prevent interferon induction
a, RECQ1, CtIP, BLM, DNA2 and WRN proteins remaining 48 h after transfection with the corresponding siRNAs (n = 2). b, SAMHD1-depleted (shSAM) HEK293T cells were transfected with the indicated siRNAs for 48 h and then treated with 4 mM HU for 2 h. Cytosolic ssDNA was visualized by immunofluorescence microscopy. Representative confocal microscopy images are shown (n = 2). Scale bar, 5 μm. c, Depletion of RecQ1 prevents the accumulation of cytosolic ssDNA in untreated shSAM cells. Cytosolic ssDNA was detected by immunofluorescence microscopy. Representative images are shown (n = 3). Scale bar, 5 μm. d, Mean fluorescence intensity of cytosolic ssDNA in the cells c was quantified by using CellProfiler (n = 130). ****P < 0.0001, Mann–Whitney rank sum test. e, RECQ1 and SAMHD1 proteins remaining after siRNA transfection, as analysed by western blotting (n = 3). f, shScr and shSAM HEK293T cells were transfected with siRNA against RECQ1 (siRECQ1) for 48 h. Expression of IFNB mRNA was quantified by qRT–PCR. Data shown are representative of three independent experiments. Error bars denote s.d. of triplicates. g, HeLa cells were transfected with siRNAs as indicated. They were cultured in the absence or presence of 4 mM HU for 8 h and then without HU for a total of 20 h before mRNA extraction. Expression of IFNA mRNA was analysed by qRT–PCR and normalized to GADPH mRNA. Data shown are representative of three independent experiments. Error bars denote s.d. of triplicates. h, shScr and shSAM HEK293T cells were transfected with siRNA against RECQ1 for 48 h. The cells were sequentially labelled for 15 min with IdU and for 15 min with CldU. Then, they were either collected immediately or treated for 2 h with 4 mM HU before DNA fibre analysis. The lengths of the IdU and CldU tracks (n = 200) were plotted as the ratio of CldU to IdU. Median values are indicated in red. ****P < 0.0001, Mann–Whitney rank sum test.
