Extended Data Fig. 3: Fork progression is altered in the absence of SAMHD1.
From: SAMHD1 acts at stalled replication forks to prevent interferon induction
a, Western blot analysis of SAMHD1 protein in SAMHD1-depleted HeLa cells (using shSAM) and in SAMHD1-depleted HEK293T cells expressing an shRNA-resistant, full-length SAMHD1 (SAM) under the control of a doxycycline-inducible promoter. Expression of SAMHD1 was analysed 72 h after induction with doxycycline. b, Control (shScr) and SAMHD1-depeleted (shSAM) HEK293T cells were seeded in 24-well plates. Cell number was determined by using trypan blue exclusion and haemocytometry. Mean and s.d. are shown from three independent experiments. c, SAMHD1 is required for normal S-phase progression. shScr and shSAM HEK293T cells were pulse-labelled with EdU for 20 min and chased with thymidine for 4 h before flow cytometry analysis. The arrowhead indicates the EdU-labelled cell population that completed DNA replication and came back to G1 phase. The percentage of cells in G2/M phase is indicated. n = 2. d, shScr and shSAM HeLa cells were labelled sequentially for 20 min with IdU and CldU and the length of CldU tracks (n = 150) was analysed by DNA fibre spreading. Median track lengths are indicated in red. e, Representative images of stretched DNA fibres. Red, IdU; green, CldU; blue, DNA. The green channel is shown separately for clarity. Scale bar, 4 µm. f, DNA fibres from shScr and shSAM HEK293T cells sequentially labelled for 20 min with IdU and CldU were either stretched on glass slides (DNA fibre spreading; n = 190) or combed on silanised coverslips (DNA combing; n = 130) and then analysed by immunofluorescence microscopy. The length distribution of CldU tracks is shown. Median track lengths are indicated in red. g, DNA fibres from cells treated as in f were stretched by DNA combing and the distance between CldU tracks, which is indicative of the density of active origins, was determined for five independent experiment. Median distances are indicated. Whiskers correspond to 10–90 percentiles. ****P < 0.0001, Mann–Whitney rank sum test. h, SAMHD1-depleted cells show increased spontaneous fork arrest. The ratio of the shortest to the longest CldU track from cells treated as in d was calculated for each pair of divergent sister replication forks and the percentage of sister forks showing a ratio of less than 0.6 is shown (n = 75). Error bars indicate s.d. from three independent experiments. P < 0.05, Mann–Whitney rank sum test.
