Fig. 5: Loss of the lncRNA ENSG00000272172.1 increases DNA damage.

a Expression of ENSG00000272172.1 in OVCAR8 cells 72 h post-transfection with either control siRNA or siRNA targeting ENSG00000272172.1, measured by real-time PCR using the ΔCt method and normalized to GAPDH. Displayed is one of three representative biological replicates. b Relative cell number in a panel of ovarian cancer cell lines (n = 6) 72 hours after transfection with control or ENSG00000272172.1-targeting siRNA, quantified using the MTS cell viability assay. Data normalized to control-siRNA-treated cells. c Quantification of EdU-positive (actively replicating) OVCAR8 cells following 72 h after siRNA-mediated knockdown of ENSG00000272172.1, assessed by immunofluorescence microscopy. Value represents percentage of positive nuclear EDU cells per field of view. d Nuclear intensities of RAD51, phosphorylated γH2AX (p-γH2AX), and RPA were quantified in OVCAR8 cells 72 h post-knockdown, using immunofluorescence and automated image analysis. e Relative survival of OVCAR8 cells treated with control-siRNA, BRCA1-siRNA, or ENSG00000272172.1-siRNA. 24 h after siRNA treatment, the cells were treated for 72 h with 5 µM or 20 µM of olaparib. Cell viability was measured by a MTS assay. f Western blot analysis of DNA damage and repair markers (p-γH2AX, 53BP1, RAD51) in OVCAR8 cells treated with control or ENSG00000272172.1-siRNA. Cells were exposed to 20 µM olaparib 24 h post-transfection and lysed after an additional 48 h. GAPDH was used as a loading control. g Quantification of RAD51-positive nuclei in OVCAR8 cells treated with control or ENSG00000272172.1-siRNA. Cells were treated with bleomycin (10 µg/mL, 1 h) 48 h post-transfection, followed by a 16-h recovery period before fixation. h Volcano plot showing differential gene expression in OVCAR8 cells following ENSG00000272172.1 knockdown versus control-siRNA treatment, based on real-time PCR profiling of a selected gene panel. Fold changes and p values were calculated using the ΔCt method. i Western blot analysis of MCM2, MCM7, CDT1 and GAPDH in OVCAR8 cells treated with control or ENSG00000272172.1-siRNA. j Western blot showing phosphorylation levels of checkpoint proteins CHK1, CHK2, and γH2AX in cells transfected with control-siRNA, BRCA1-siRNA, or ENSG00000272172.1-siRNA. At 48 h post-transfection, cells were treated with 2 mM hydroxyurea (HU) for 1 h before lysis. For all quantified assays, results are representative of at least three biological replicates unless otherwise noted. Statistical analysis was performed using unpaired two-tailed Student’s t-tests. Significance is indicated as p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), and p < 0.0001 (****).