Fig. 3: COL4A6 regulates DDR1 transcription by the upregulation of E2F1 binding to the DDR1 promoter, and COL4A6 regulates cell invasion ability via the E2F1/DDR1/FAK axis.

A The protein expression levels of COL4A6 and DDR1 in a panel of ovarian cancer cells were evaluated by western blotting. β-actin served as a protein loading control. All experiments were performed in triplicate. B The mRNA expression levels of COL4A6 and DDR1 in OVCAR-4 and A2780 cells transfected with different quantities of a COL4A6 plasmid (1 and 3 µg) were evaluated by qRT-PCR. All experiments were performed in triplicate. C The mRNA-expression levels of COL4A6 and DDR1 in OVCAR-8 and A2780CP70 cells transfected with different quantities of a COL4A6 knockdown plasmid (1 and 3 µg) were evaluated by qRT-PCR. All experiments were performed in triplicate. D Schematic diagram of the DDR1 promoter-driven luciferase reporter: DDR1 (–180/ + 1). E2F1 has a putative E2F1-binding site. The promoter constructs were co-transfected into A2780, OVCAR-8, OVCAR-4, and A2780 cells, and luciferase reporter assays were performed 48 h later. The one putative E2F1-binding site on DDR1 (–180/ + 1) was changed by site-directed mutagenesis, and the mutated promoters were transfected into OVCAR-8 and OVCAR-4 cells to evaluate reporter activity. (E) Upper panel: Western blotting was performed to evaluate COL4A6, E2F1, DDR1, p-DDR1, FAK, and p-FAK protein expression in COL4A6 knockdown OVCAR-8 and COL4A6-overexpressing OVCAR-4 cells. Lower panel: The binding of E2F1 to the DDR1 promoter was evaluated by ChIP in COL4A6 knockdown OVCAR-8 and COL4A6-overexpressing OVCAR-4 cells. Chromatin was isolated and immunoprecipitated using an anti-E2F1 antibody. F Upper panel: The binding of E2F1 to the DDR1 promoter was evaluated by ChIP in OVCAR-8 cells transiently transfected with different quantities of a COL4A6 knockdown plasmid (1 and 3 µg) and an E2F1 cDNA plasmid (3 µg), and OVCAR-4 cells were transfected with different quantities of a COL4A6 plasmid (1 and 3 µg) and an E2F1 knockdown plasmid (3 µg). Chromatin was isolated and immunoprecipitated using an anti-E2F1 antibody. Lower panel: OVCAR-8 cells were transfected with a COL4A6 knockdown plasmid and an E2F1 cDNA plasmid, and OVCAR-4 cells were transfected with a COL4A6 cDNA plasmid and E2F1 knockdown plasmid. DDR1 mRNA levels and promoter activities were evaluated by performing qRT-PCR and luciferase assays, respectively. All experiments were performed in triplicate. P values were determined using the Student’s t-test. ^*P < 0.05 or ^**P < 0.005^, relative to the control cells treated with shV or VC. G Left panel: OVCAR-8 cells were transiently transfected with COL4A6 knockdown plasmid and a DDR1 cDNA plasmid. After 48 h, COL4A6, DDR1, p-DDR1, FAK, and p-FAK expressions were evaluated by western blotting in whole lysates after both treatment conditions. β-actin was used as a protein loading control. Right panel: The cells were trypsinized and collected from the dishes. Samples consisting of 1 × 10⁴ cells were seeded into Transwells to evaluate invasion capacity. All data represent the means ± standard deviations of three separate experiments. (H) Left panel: OVCAR-4 cells were transiently transfected with COL4A6-expression plasmid and a DDR1 knockdown plasmid. After 48 h, COL4A6, DDR1, p-DDR1, FAK, and p-FAK expression was evaluated by western blotting in whole lysates after both treatment conditions. β-actin was used as a protein loading control. Right panel: The cells were trypsinized and collected from the dishes. Samples consisting of 1 × 10⁴ cells were seeded into Transwells to evaluate invasion capacity. All data represent the means ± the standard deviations of three separate experiments. I Left panel: western blotting was performed to evaluate COL4A6, DDR1, p-DDR1, FAK, and p-FAK protein expression after treatment of A2780-COL4A6 expression cells for 24 h with FAK inhibitor. β–actin was the loading control. Right panel: In vitro invasion activity of A2780-COL4A6 expression cells after treatments. All data represent the mean ± the standard deviation of three separate experiments.