The human ovarian granulosa cell line KGN was obtained from the RIKEN BioResource Center. Cells were cultured in DMEM/F12 medium (Gibco, USA) containing 10% foetal bovine serum (FBS) (HyClone, USA) and 1.0% penicillin/streptomycin (PS) (Gibco, USA). KGN cells were seeded in 60-mm plates (1×106 cells/plates) and cultured at 37°C with 5% CO2 until the cells adhered to the wall. Cultured cells were divided into six groups: (1) control group, in which granulosa cells were cultured in DMEM without any treatment for 24 h; (2) 10− 11 M T group, in which granulosa cells were cultured in DMEM with 10− 11 M T (Sigma, USA) for 24 h; (3) 10− 7 M T group, in which granulosa cells were cultured in DMEM with 10− 7 M T for 24 h; (4) 10− 5 M T group, in which granulosa cells were cultured in DMEM with 10− 5 M T for 24 h; (5) 10− 7 M T + flutamide group, in which granulosa cells were cultured in DMEM with 10− 7 M T for 24 h after the 10− 6 M flutamide (Sigma, USA) treatment for 1 h; (6) 10− 7 M T + GF109203X group, in which granulosa cells were cultured in DMEM with 10− 7 M T for 24 h after the 10− 6 M GF109203X (MCE, USA) treatment for 1 h. The follow-up experiment was performed immediately following the culture.
After the medium was discarded, the cells were washed twice with phosphate-buffered saline (PBS). Cells were lysed for 20 min in RIPA buffer with protease inhibitors (100:1) and then centrifuged at 12000 r/min at 4°C for 5 min. The supernatant was transferred to a new centrifuge tube for Western blot analysis. After determination of the protein content by the bicinchoninic acid (BCA) protein assay, 25 µg proteins were separated by 10% SDS-PAGE and transferred onto nitrocellulose membranes. After the membranes were blocked with 5% skim milk for 1 h, they were incubated with Cx43 antibody (1:1000, CST, USA), phosphorylated connexin 43 (p-Cx43) antibody (1:1000, CST, USA), and PKC antibody (1:1000, Abcam, UK) for 1 h at room temperature and overnight at 4°C. After three washes in Tris-buffered saline (TBS)-0.1% Tween 20, the membranes were incubated with goat anti-rabbit IgG and goat anti-mouse IgG conjugated with horseradish peroxidase (HRP) (1:5000, Proteintech Group, China) for 1 hour at room temperature. The membranes were washed again as described above, and the immunoreactivity was examined by an enhanced chemiluminescence (ECL) system. As a control for normalization, the membranes were reprobed for the internal control GAPDH (1:5000, Atagenix, China). Films were scanned, and the optical density of the bands was measured with AlphaEaseFC. The relative quantities of Cx43, p-Cx43, and PKC were determined with reference to GAPDH. The final data are expressed as the mean of the results of three independent experiments performed at different time points.
KGN cells were seeded on coverslips in 60-mm plates and cultured as described above. After three washes with PBS, the cells were fixed in 4% paraformaldehyde for 15 min and blocked with goat serum for 30 min at room temperature. After the supernatant was discarded, the cells were incubated overnight with Cx43 antibody (1:1000, CST, USA) or p-Cx43 antibody (1:1000, CST, USA) at 4°C. After three washes, the cells were incubated with goat anti-rabbit IgG secondary antibody (1:5000, Proteintech Group, China) for 1 h at room temperature. DAPI was dropped on coverslips, incubated for 5 min in the dark, and then viewed and imaged under a fluorescence microscope.
Scrape loading/dye transfer
GJIC was determined according to the Scrape loading/dye transfer method described previously[13–15], which used Lucifer yellow dye that can diffuse through gap junctions. KGN cells were cultured as described above, and after the medium was discarded, the cells were washed three times with preheated PBS at 37°C. Two millilitres of 0.05% Lucifer yellow dye preheated at 37°C was added to the culture dishes. The cell layer was gently cut with a sterile scalpel and then incubated for 3 min under dark conditions. After incubation, the cells were washed with PBS three times, fixed with 4% paraformaldehyde, viewed and imaged immediately under a fluorescence microscope. GJIC activity was defined as the distance that the Lucifer yellow dye transferred from the scrape line. Values are expressed as a fraction compared to the control.
All analyses were performed with Software Package for Social Sciences (SPSS) version 10.0 for Windows. All data are expressed as the mean ± standard deviation (SD). Comparisons among multiple samples were performed with variance analysis, and intergroup multiple comparisons were performed with Bonferroni tests. P < 0.05 was considered statistically significant.