Fig. 4: EWS::FLI1-DHX9 associated R-loop accumulation is a source of replication stress.

A Analysis of the effect of RNH1 overexpression in SN-38-induced replication stress by pCHK1 (Ser345) IF. Cells were transfected with RNH1:GFP or control plasmids 24 h before 5 µM SN-38 treatment (30 min). Left, representative images. DAPI counterstain. Scale bar, 20 µm. Right, data represent the mean (±SEM) of pCHK1 intensity in GFP-positive cells, n ≥ 2 independent experiments (30 cells were analyzed per replicate). B Effect of EWSR1::FLI1 knockdown in SN-38-induced replication stress by pCHK1 (Ser345) WB. shA673 cells were pre-incubated with DOX and treated with 5 µM SN-38 (30 min). Loading control: GAPDH. Molecular weight in kDa. Asterisk indicates non-specific bands. Bottom, quantification of CHK1 phosphorylation (pCHK1/CHK1 band signal). Data represent the mean of 2 independent experiments (relative to SN-38). C Similar to (B), by DNA fiber assay. Cells were incubated with CidU (30 min) and IdU + 5 µM SN-38 (30 min). D Quantification of (C). Data represent the mean (±SEM) of IdU/CidU fiber length ratio, n = 2 independent experiments (more than 250 fibers were analyzed per condition). E Effect of DHX9 overexpression on SN-38-induced replication stress by pCHK1 (Ser345) WB. A-673 and TC-71 cells overexpressing DHX9:GFP or control plasmids were treated with 5 µM SN-38 (30 min). Bottom, quantification of CHK1 phosphorylation (pCHK1/CHK1 band signal). Data are the mean of 2 independent experiments (relative to SN-38 GFP). Other details as in (B). F Effect of YK-4-279 treatment on SN-38-induced replication stress by pCHK1 (Ser345) WB. Cells were pre-incubated with 75 µM YK-4-279 (1.5 h) and treated with 5 µM SN-38 (30 min). Other details as in (B). G Evaluation of the effect of ATR inhibition in SN-38-induced pCHK1 and γH2AX by WB. A-673 and TC-71 cells were pre-incubated with 10 µM AZD6738 for 24 h and treated with 5 µM SN-38 (30 min). Other details as in (B). H Analysis of the effect of ATR inhibition in SN-38-induced chromosomal breaks. Cells were pre-incubated with 10 µM AZD6738 for 24 h and treated with 2.5 µM SN-38 (A-673) or 1.25 µM SN-38 (TC-71) for 30 min. Data represent the mean (±SEM) of the frequency of chromosomal breaks per chromosome, n ≥ 2 independent experiments (20 metaphases were analyzed per replicate). Statistical significance was determined by t-test. I Left, synergy plot representing synergistic effect of AZD6738 and SN-38 combination in A-673 cells. Right, effect of ATR inhibition in cell survival upon SN-38 exposure by MTT assay. Cells were incubated with AZD6738 (250 nM, 24 h) previous to the treatment with indicated concentrations of SN-38 (72 h). Data represent the mean (+SEM) of the percentage of survival, n = 3 independent experiments. Dotted lines indicate IC50. J Similar to (I) in TC-71 cell line. Statistical significance was determined by t-test.