Fig 1: R-loops slow replication rate in INO80-depleted cells.a Schematic representation of the experimental approach. PC3 cells were transfected with esiRNAs against either GFP or INO80. Three days later, cells were treated with α-amanitin (α-a) or cordycepin (crd) for 3 h or left untreated as control and then subjected to fibre labelling analysis. b Representative images of spread fibres from each condition. Similar results were obtained in five independent experiments (cordycepin treatment—2). c Distribution of fork speed rates in INO80-proficient (siGFP) and INO80-deficient (siINO80). Data is from five independent experiments (in cordycepin-treated cells—2), at least 250 fibres were measured per condition in each experiment. ****p-value < 0.0001, (two- tailed unpaired Student’s t-test). d Schematic representation of the experimental setup used. PC3 cells were co-transfected with esiRNAs against either GFP (siGFP) or INO80 (siINO80) along with either a control (CTRL) or RNase H1-overexpressing (RNAseH1) vector. Two days later RNAse H1 expression was induced by doxycycline for 24 h. Cells were labelled with CldU for 5 min followed by IdU pulse for 20 min and subjected to DNA fibre labelling analysis. e Representative images of spread fibres from each condition. Similar results were obtained in four independent experiments. f Distribution of fork speed rates (kilobase/min) in siGFP and INO80-deficient cells transfected with control or RNAse H1 overexpression plasmids. Data are from 4 independent experiments, at least 250 fibres were measured per condition in each experiment. ****p-value < 0.0001, (two-tailed unpaired Student’s t-test). g Schematic representation of the experimental setup. Cells were co-transfected and induced as in d and prior to labelling were treated with 5 µM vorinostat for 6 h. h Representative images of spread fibres from each condition. Similar results were obtained in three independent experiments. i Distribution of fork rates from (h, at least 250 fibres were measured per condition in each experiment; ns non-significant, ****p-value < 0.0001, *p-value < 0.05, (two-tailed unpaired Student’s t-test). c, f, i Kruskal–Wallis test p-value was < 0.0001. Data is presented as Tukey boxplot (box representing first quartile, median and third quartile, whiskers 1.5 times interquartile range).
Fig 2: Replication stress-induced DNA damage in INO80-deficient cells is caused by R-loops.a PC3 cells were transfected with control vector (CTRL) and either esiRNA against GFP (lane 1) or INO80 (lane 2) or transfected with RNAse H1-expressing plasmid (RNAseH1) and esiRNA against GFP (lane 3) or INO80 (lane 4). Forty-eight hours later cells were induced with doxycycline and 24 h later analyzed by Western with an antibody against pChk1-Ser345. Similar results were obtained in two independent experiments. b Schematic representation of the experimental setup used. PC3 cells were co-transfected with esiRNAs against either GFP (siGFP) or INO80 (siINO80) along with either a control (CTRL) or RNAse H1-overexpressing (RNAseH1) vector. Two days later RNase H1 overexpression was induced by doxycycline for 24 h. To distinguish cells in S-phase, cells were labelled with 25 µM EdU, fixed and stained with an antibody against γH2AX and “clicked” with Alexa Fluor 488 azide. c Representative images of cells as in b. Scale bar 10 µm d Distribution of nuclear γH2AX staining intensities in S-phase cells; ****p-value < 0.0001, ns nonsignificant (two-tailed unpaired Student’s t-test). At least 500 cells were measured per condition in each of three independent experiments. Data is presented as Tukey boxplot. e Distribution of nuclear γH2AX staining intensities in non-S-phase cells. Data in d, e are from three independent experiments following normalization (to median intensity of entire population of siGFP/CTRL sample in each experiment). Tukey boxplot is used. Source data are provided as a Source data file.
Supplier Page from DNASU for RNASEH1P1 (Homo sapiens) in pDONR201 (Gateway donor/master vector)