Fig. 3: Loss of EWS::FLI1-DHX9 interaction alleviates R-loops accumulation promoting drug resistance.

A Kaplan–Meier showing overall survival of 166 EwS patient samples stratified by DHX9 mRNA levels. Statistical significance was determined by the Mantel–Cox test. B Determination of R-loops-DHX9 interactions by PLA in shA673 cells upon DOX incubation, and C DOX incubation and 5 µM SN-38 treatment (30 min). Left, representative images (red, PLA foci; blue, DAPI counterstain). Scale bar, 20 µm. Right, data represent the mean (±SEM) of PLA foci, n = 3 independent experiments (40 (B) and 20 (C) cells were analyzed per replicate). D Evaluation of the effect of DHX9 downregulation on SN-38-induced DSBs upon EWS::FLI1 depletion by γH2AX IF. After 48 h of transfection with indicated siRNAs and/or 24 h of incubation with DOX, shA673 cells were treated with 5 µM SN-38 (30 min). Data represent the mean (±SEM) of nuclear γH2AX signal; n = 3 independent experiments (150 cells were analyzed per replicate). E Evaluation of the effect of DHX9 overexpression on SN-38-induced DSBs by γH2AX IF. A-673 and TC-71 cells overexpressing DHX9:GFP or control plasmids were 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 signal of GFP-positive cells, n ≥ 3 independent experiments (30 cells were analyzed per replicate). F Effect of DHX9 overexpression on SN-38-induced apoptosis by AnnexinV FACS. Cells overexpressing DHX9:GFP or control plasmids were treated with 5 µM SN-38 (3 h). After washout, cells were maintained in drug-free medium for 24 h. Data represent the mean (+SEM) of the percentage of AnnexinV-positive between GFP-positive cells (relative to SN-38-), n ≥ 3 independent experiments. G Evaluation of the association between SN-38 activity (AUC) and DHX9 expression levels in EwS cell lines (n = 16). Lineal correlation was determined by the Pearson correlation coefficient. H Analysis of the effect of YK-4-279 on SN-38-induced DSBs by γH2AX IF. A-673 cells were pre-incubated with 75 µM YK-4-279 (1.5 h) and treated with 5 µM SN-38 (30 min). Left, representative images. DAPI counterstain. Scale bar, 50 µm. Right, data represent the mean (±SEM) of nuclear γH2AX intensity, n = 3 independent experiments (30 cells were analyzed per replicate). P-value was determined by t-test.