Figure 5A Changes in WT and variant FOXL2 mRNA levels in KGN cells were assessed by real-time RT-PCR after transfecting siRNAs against the indicated factors for 48 h. The data are presented as the mean ± SEM from three independent experiments, performed in triplicate.B FOXL2 protein-expression levels were determined by western blotting after transfecting KGN cells with control, DHX9, or GW182 siRNAs for 48 h. Quantification of FOXL2 protein expression is presented in the bottom panel. The data are presented as the mean ± SEM from three independent experiments. The p values were analyzed by unpaired, two-tailed Student's t-test (**p < 0.01, ***p < 0.001).C Relative binding affinities of DHX9 and GW182 to AGOs. 293T cells were transfected with expression vectors encoding the indicated FLAG/HA-tagged AGOs, and cell extracts were prepared and immunoprecipitated with an anti-FLAG antibody, followed by immunoblot analyses (top). The empty p3XFLAG-CMV-10 vector was used as a control. The band intensities of immunoprecipitated DHX9 and GW182 were quantified and normalized following pull-down with the indicated AGOs (bottom). The data are presented as the mean ± SEM from three independent experiments. * and # indicate statistically significant differences in the respective amounts of DHX9 or GW182 bound to AGO1. The p values were analysed by unpaired, two-tailed Student's t-test (p < 0.05).D Association of endogenous miR-1236 with RISC components in KGN cells was determined via pulldown assays using immobilized 2′-O-methylated oligonucleotides (2′-O-Me oligos) complementary to miR-1236 followed with a pulldown using streptavidin-coupled Dynabeads and western blot analyses (top). Relative quantification of bound proteins compared with proteins from the input is presented as fold enrichment (bottom). Efficient pulldown of endogenous miR-1236 using the 2′-O-Me oligos was confirmed with depleted miR-1236 in the discarded supernatant following the pulldown (Appendix Fig S8). As a control, 2′-O-Me oligos not complementary to miR-1236 were used. The data are the means ± SEM from three independent experiments. The p values were analyzed by unpaired, two-tailed Student's t-test (*p < 0.05, **p < 0.01).E Following transfection of control siRNA or siDHX9 into KGN cells, AGO3-mediated RISC-associated RNAs were immunoprecipitated using an anti-AGO3 antibody. IgG was used as a control. Co-immunoprecipitated mRNAs were reverse transcribed using a FOXL2-430-R primer binding downstream of the 402C>G site. The cDNA products were used for FOXL2 allele-specific PCR analysis with a FOXL2-279F primer (Appendix Fig S1A), and a representative result obtained by RT-PCR (top) is shown. Quantitative real-time RT-PCR results (middle) are also presented as fold enrichment of FOXL2 mRNAs normalized using the level of GAPDH mRNA. Western blots of immunoprecipitated AGO3 and the inputs are shown in the bottom panel. The data are presented as the mean ± SEM from three independent experiments. Different letters denote statistically significant differences (p < 0.05; Student-Newman-Keuls test).F We examined whether DHX9 affected the association between miR-1236 and AGOs. After transfecting KGN cells with control siRNA or siDHX9, the total RNA and AGOs-mediated RISC-associated RNAs were isolated following immunoprecipitations using anti-AGO3 or anti-AGO2 antibodies. The AGOs-immunoprecipitated RNAs were extracted using an acidic phenol:chloroform mixture (5:1, pH 4.3) and precipitated with isopropanol using 10% of 3 M NaOAc (pH 5.2). The enrichment of miR-1236 within miRISCs was detected using the TaqMan® microRNA assay in the immunoprecipitated RNAs and normalized using the level of total miR-1236. The data (means ± SEM) are presented as the fold enrichment calculated from three independent experiments. Different letters denote statistically significant differences (p < 0.05; Student-Newman-Keuls test).G, H Luciferase activities of the reporter constructs presented in Fig 3A and C were measured in KGN cells after transfecting the miR-1236 mimic, indicated siRNAs, and either pGL3c-CDS-FOXL2 MT or pGL3c-UTR-FOXL2 MT for 48 h. The data are expressed as the means ± SEM from three independent experiments and were performed in triplicate. The p values were analyzed by unpaired, two-tailed Student's t-test (***p < 0.001).