Fig. 3: FHL2 deficiency inhibited GC viability and EGF-induced GC proliferation in vitro.

A Fhl2 mRNA levels in GCs transfected with scramble siRNA (Ctrl) or Fhl2 specific siRNA (siFHL2). N = 3 for each group. β-actin was used as a loading control. B Knockdown of FHL2 inhibited cell viability significantly. Cell viability was detected by CCK-8. N = 3 for each group. C Quantitative data showing cell number changes in control and FHL2 knockdown cells. N = 3 for each group. D Flow cytometry showing cell cycle distribution in GCs transfected with scramble siRNA (Ctrl) or Fhl2 specific siRNA (siFHL2). Data was displayed as a percentage of cells in each phase of the cell cycle. N = 3 for each group. E Fhl2 mRNA levels in GCs transfected with control vector (Ctrl) or Fhl2 overexpression vector (Fhl2 O/E). N = 3 for each group. β-actin was used as loading control. F Overexpression of FHL2 promoted GC viability. N = 4 for each group. G Cell growth changes in control and FHL2 overexpression cells. N = 3 for each group. H Overexpression of FHL2 dramatically deceased cell apoptosis. Cells were stained with an Annexin V-APC/PI dual staining kit and apoptosis was analyzed by flow cytometry. N = 3 for each group. I EGF treatment improved cell viability in a dose-dependent manner. GCs were incubated in medium containing DMEM/F12 (1:1) and 1% fetal bovine serum (FBS) in the absence (vehicle control, Ctrl) or presence of 1, 10, or 20 ng/ml EGF for 48 h. CCK-8 was employed to measure GC viability. N = 3 for each group. J EGF treatment stimulated GC proliferation. GCs were incubated in the absence (vehicle control, Ctrl) or presence of EGF (20 ng/ml) in medium containing DMEM/F12 (1:1) and 1% FBS for 48 h. N = 3 for each group. K Cell cycle distribution in GCs treated with absence or presence of EGF. N = 3 for each group. Cell cycle analysis was performed by flow cytometry. L Knockdown of FHL2 attenuated EGF induced cell proliferation. GCs were transfected with scramble siRNA (Ctrl) or Fhl2 specific siRNA (siFHL2) for 24 h, followed by EGF treatment (20 ng/ml) for 48 h. Cell viability was measured by CCK-8. N = 4 for each group. M Knockdown of FHL2 in vitro decreased protein levels related with cell growth. GCs were transfected with either scramble siRNA (Ctrl) or Fhl2 specific siRNA (siFHL2) for 72 h. Protein levels were determined by western blot. GAPDH was used as a loading control. All the experiments were repeated at less 3 times and representative images were presented. N Representative western blot images showing the protein expressions in wildtype (WT) and Fhl2 KO mice ovaries. Whole ovary extracts were prepared for western blot. β-actin was used as the loading control. All the experiments were repeated at less 3 times. Student’s t test or one-way ANOVA were used to compare the difference between groups. Data are shown as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns non-significant.