Fig. 2: EWS::FLI1 impairs drug-induced R-loop resolution promoting genome instability and cytotoxicity.

A Evaluation of the association between CPT and B SN-38 activity (AUC), and EWSR1::FLI1 expression levels in EwS cell lines (n = 16). FLI1 gene was used as a surrogate marker of EWSR1::FLI1, since FLI1 is not expressed in EwS cells. Lineal correlation was determined by Pearson correlation coefficient. C EWS::FLI1 and LOX protein levels by WB in shA673 cells after DOX incubation. Loading control: GAPDH. Molecular weight in kDa. D SN-38-induced apoptosis upon EWSR1::FLI1 knockdown by PARP1 and CASP3 WB. Cells were pre-incubated with DOX for 24 h before 5 µM SN-38 treatment (3 h). After washout, cells were cultured in drug-DOX-free medium for 24 h. Loading control: GAPDH. Molecular weight in kDa. E Similar to (D) by AnnexinV FACS. Data represent the mean (+SEM) of the percentage of AnnexinV-positive cells, n = 5 independent experiments. F Similar to (D) by MTT assay. Cells were treated with indicated concentrations of SN-38 (3 h). After treatment, cells were cultured in drug-DOX-free medium for 48 h. Data represent the mean (+SEM) of the percentage of survival (relative to DMSO), n = 5 independent experiments. Dotted lines indicate IC50. G SN-38-induced γH2AX upon EWSR1::FLI1 knockdown by IF. Cells were pre-incubated with DOX for 24 h and treated with 5 µM SN-38 (30 min). Left, representative images. DAPI counterstain. Scale bar, 20 µm. Right, data represent the mean (±SEM) of nuclear γH2AX intensity, n = 5 independent experiments (~100 cells were analyzed per replicate). H SN-38-induced chromosomal breaks upon EWSR1::FLI1 downregulation. After DOX incubation, cells were treated with 2.5 µM SN-38 (30 min). Left, representative image. Scale bar, 10 µm. The arrow indicates a chromosomal break. Right, data represent the mean (±SEM) of the frequency of chromosomal breaks per chromosome, n = 3 independent experiments (20 metaphases were analyzed per replicate). I EWS::FLI1 protein levels in HeLa EF cells after 72 h of DOX incubation. Loading control: GAPDH. Molecular weight in kDa. J SN-38-induced γH2AX upon EWSR1::FLI1 overexpression by IF. HeLa EF cells were pre-incubated with DOX for 72 h and treated with 5 µM SN-38 (30 min). Other details as in (G), n = 3 independent experiments (~100 cells were analyzed per replicate). K SN-38-induced R-loops upon EWSR1::FLI1 knockdown by s9.6 IF. Cells were pre-incubated with DOX and treated with 5 µM SN-38 (30 min). Left, representative images. DAPI counterstain. Scale bar, 20 µm. Right, data represent the mean (±SEM) of nuclear s9.6 intensity, n = 3 independent experiments (~75 cells were analyzed per replicate). L Effect of RNH1 overexpression on SN-38-induced DSBs. Cells were transfected with RNH1:GFP or control plasmids 24 h before treatment with 5 µM SN-38 (30 min). Left, representative images. DAPI counterstain. Scale bar, 20 µm. Right, data represent the mean (±SEM) of nuclear γH2AX intensity in GFP-positive cells, n = 3 independent experiments (~50 cells were analyzed per replicate). P-value was determined by t-test.