Fig. 4

Effects of JAK3 inhibition or overexpression and FSH treatment on STAT3 phosphorylation in granulosa cells. a To analyse the effects of JAK3 manipulations on downstream effectors in GC, Western blot analyses were performed and revealed phosphorylation status of STAT3 following different treatments. JAK3 was overexpressed in GC using the pQE system (+ JAK3) with or without JNX treatment for 8 or 24 h to inhibit JAK3 phosphorylation. Detected bands and their relative abundances were quantified and phosphorylation status of STAT3 (pSTAT3) was analysed. A significant increase in pSTAT3 amounts was observed when JAK3 was overexpressed. JNX treatment for 8 h resulted in a significant decrease in pSTAT3. Overexpression of JAK3 following 8 h JNX treatment restored pSTAT3 amounts to the control levels. However, overexpression of JAK3 was not able to restore or increase pSTAT3 within GC after 24 h of JNX treatment, which significantly decreased pSTAT3 as compared to the control group and JAK3-overexpressed cells (red box). b and c Further experiments allowed to evaluate the effects of FSH administration on the phosphorylation status of STAT3 in cultured GC. Fold changes of pSTAT3 over to total STAT3 were compared following treatments with JNX (24 h), FSH (4 h) or JAK3 overexpression. JNX significantly reduced phosphorylation of STAT3 after 24 h of treatment, while overexpression of JAK3 without JNX treatment significantly increased the amount of pSTAT3 within transfected GC similar to the result in A. Addition of FSH significantly increased pSTAT3 compared to the JNX group. Moreover, combination of JAK3 overexpression with FSH treatment increased pSTAT3 but not to the same levels as either FSH alone or JAK3 overexpression alone. Addition of FSH in the presence of JNX weakened the negative effect of JNX on pSTAT3 as compared to JAK3 overexpression in the presence of JNX (blue bar). CTRL, control; FSH, Follicle-stimulating hormone; JNX, JANEX-1; + JAK3, JAK3 overexpression. Different letters denote significant differences among samples (ANOVA, Tukey–Kramer multiple comparison)