Figure 4

PERK/NRF2 pathway activation was responsible for increased CX43 and StAR-dependent progesterone synthesis in the ovary of female mice under cold exposure. (A) Potential cis-acting elements matching to several transcriptional factors (NRF2, STAT3, AP-1 and MyoD) were identified within mouse CX43 promoter. (B) Female mice were untreated or exposed to cold stress as described in Fig. 1A. The ovaries of the mice in each group were collected and subjected to western-blot assay to detect the expression or activation levels of the above transcriptional factors. (C) The quantitative results of NRF2 activation (p-NRF2 relative to total NRF2) were shown (**p < 0.01). (D) The samples in B were used to detect the activation status of PERK. (E) The quantitative results of PERK activation (p-PERK relative to total PERK) were shown (**p < 0.01). (F,G) The primary granulosa cells (GCs) were extracted and treated as described in Fig. 2E,G. Then the activation status of PERK and NRF2 were detected. (H and K) The primary GCs were transfected with NRF2 siRNA or their control siRNAs, followed by exposure to cold stress for 12 h. Then the changes on CX43 and StAR expression (H) and the levels of progesterone in cell culture supernatants (K) were detected (**p < 0.01, *p < 0.05). (I,L) The primary GCs were transfected with PERK siRNA or their control siRNAs, followed by exposure to cold stress for 12 h. Then the changes on PERK/NRF2/CX43/StAR pathway activation (I) and the levels of progesterone in cell culture supernatants (L) were detected (**p < 0.01, *p < 0.05). (J) The primary GCs were pre-treated with PERK inhibitor GSK2606414 or its solvent DMSO, followed by exposure to cold stress for 12 h. Then the changes on PERK/NRF2/CX43/StAR pathway activation were detected.