parental es2  (ATCC)

Journal: bioRxiv

Article Title: The miR-221-5p/RAD18/RAD51 Axis Regulates DNA Damage Tolerance and Homologous Recombination to Drive Platinum Resistance in Ovarian Cancer

doi: 10.64898/2026.05.11.724004

Figure Lengend Snippet: (A–F) Western blot analyses of RAD18 and RAD51 in OVCAR8 (A,B), ES2 (C,D) and SKOV3 (E,F) cells 48h after transfection with a control miR or a miR–221–5p mimic. Vinculin serves as loading control. Corresponding bar graphs in B, D and F show densitometric quantification of protein expression normalized to vinculin (mean ± SD, n = 3 independent replicates; ****P < 0.0001, two–tailed t -test). (G–I) Functional rescue experiment in OVCAR8 cells. Cells were transfected with control miR, miR–221–5p mimic or a miR–221–5p inhibitor (anti-miR). Immunoblot blot (G) and quantification (H, I) demonstrate that miR–221–5p-mediate downregulation of RAD18 and RAD51 effectively reversed by the inhibitor, confirming the specificity of this regulatory axis. (J) Schematic representation of firefly luciferase reporter constructs containing the 3′–untranslated regions (3′ UTRs) of RAD51 or RAD18 . Conserved miR–221–5p seed sequences within each 3′ UTR are highlighted. (K) Relative luciferase activity in OVCAR8 and ES2 cells co-transfected with miR-221-5p mimics and the indicated 3′ UTR reporter vectors. Values were normalized to internal controls. Data are presented as mean ± SD of n = 3; *P < 0.05, **P < 0.01, ***P < 0.001 by two–tailed t-test).

Article Snippet: The human epithelial ovarian cancer (OC) cell lines OVCAR3, OVCAR4, OVCAR8, OV90, ES2, A2780, and SKOV3 were purchased from the American Type Culture Collection (ATCC; Manassas, VA).

Techniques: Western Blot, Transfection, Control, Expressing, Two Tailed Test, Functional Assay, Luciferase, Construct, Activity Assay

Journal: bioRxiv

Article Title: The miR-221-5p/RAD18/RAD51 Axis Regulates DNA Damage Tolerance and Homologous Recombination to Drive Platinum Resistance in Ovarian Cancer

doi: 10.64898/2026.05.11.724004

Figure Lengend Snippet: (A–C) Western blot analysis of OVCAR8 (A), ES2 (B) and SKOV3 (C) cells were transfected with a control miR or a miR-221-5p mimic and exposed to carboplatin (Carbo, 300 μM) for 24h. Total PCNA and GAPDH serve as loading controls. (D) OVCAR8 cells were transfected with pDR-GFP and selected using 5 µg/ml puromycin. Stably expressing cells were simultaneously transfected with miR-control or miR-221-5p mimics along with pCBASceI expression vector. After 48 h post-transfection, the GFP-positive cells was quantified via flow cytometry (BD Accuri) to determine the relative HR efficiency (mean ± SD of three independent experiments; ****P < 0.0001; two-tailed t -test). (E) Alkaline comet assay images of SKOV3 cells transfected with control miR or miR-221-5p mimic and treated with carboplatin (100 μM) for vehicle for 24h or vehicle. (F) Quantification of comet tail area from (E). Each point represents an individual nucleus; horizontal bars denote the mean ± SD (more than 25 cells per condition). (***P < 0.001, ****P < 0.0001; two-tailed t -test; ns, not significant).

Article Snippet: The human epithelial ovarian cancer (OC) cell lines OVCAR3, OVCAR4, OVCAR8, OV90, ES2, A2780, and SKOV3 were purchased from the American Type Culture Collection (ATCC; Manassas, VA).

Techniques: Western Blot, Transfection, Control, Stable Transfection, Expressing, Plasmid Preparation, Flow Cytometry, Two Tailed Test, Alkaline Single Cell Gel Electrophoresis