Fig. 1.

Effects of neuregulin-1 (NRG1) knockdown in granulosa cells (GCs). Pregnant Mare Serum Gonadotropin (PMSG) primed GCs were isolated from two ovaries per rat per sample and grown in culture dishes. GCs were transiently transfected with small-interfering-NRG1 (siNRG1) RNA and scramble-green-fluorescent-protein (GFP) RNA (negative control). Thereafter, cells were maintained in serum-free media and harvested at 24 and 48 h. A Representative images of post-transfection live-cell phase (Bright field) and green-fluorescence (scramble) show the transfection efficiency in GCs. B and D NRG1 and selective cytokines (interleukin (IL)-1α, IL-1β, IL-6, IL-8, IL-10 and tumor necrosis factor-α (TNFα) mRNA expression levels were detected by real-time qPCR in the scramble and siNRG1 transfected group. The levels of mRNA were normalized to Glyceraldehyde 3-phosphate dehydrogenase (GAPDH). C Representative immunoblots showing NRG1 and ErbB3 protein expression in GCs. Equal amounts of protein were applied to each lane. β-actin was used as an internal control. All bar graphs represent the mean ± SEM of results from three individual experiments (n = 3). Bar diagrams for protein represent the densitometric analyses of NRG1 and ErbB3 of immunoblots. Asterisks (*) represent unpaired Student t-test, *p ≤ 0.01, **p ≤ 0.001, ***p ≤ 0.0001, NS-not significant