Fig 2: Morphological changes between SWFs and ASWFs. (A) The expression of FSHR in SWFs or ASWFs. Scale bar: 20 µm. GL: Granulosa layer; TL: Theca layer. (B) β-galactosidase staining of SWFs or ASWFs. Scale bar: 50 µm. (C) The expression of γH2AX in SWFs or ASWFs. Scale bar: 20 µm. (D) The expression of CDKN1A in SWFs or ASWFs. Scale bar: 20 µm. (E) The expression of CHK2 in SWFs or ASWFs. Scale bar: 20 µm. (F) The expression of p-CHK2 in SWFs or ASWFs. Scale bar: 20 µm. (G) The ultrastructure of SWFs or ASWFs in hens aged 280 days. Scale bar: 1 µm (left) or 500 nm (right). (H) Relative expression of proteins related to DNA damage in the follicles. * p < 0.05, ** p < 0.01, *** p < 0.001.

Fig 3: Effect of FSH on DNA damage of cultured GCs via CHK2 activation. GCs were treated with 0.01 IU/mL FSH and/or 50 μM ACNU for 24 h. (A) Changes in cell viability using the CCK-8 assay. Values are the means ± SEM (n = 3). Bars with different superscripts are statistically different (p < 0.05). (B) Relative expression of proteins and mRNA related to DNA damage in the follicles. Bars with different superscripts are statistically different (p < 0.05). (C) The expression of CHK2 in GCs after treatment of ACNU and/or FSH. Scale bar: 20 µm.

Fig 4: The interactions of CHK2 with FoxK or FoxO3a. (A) The cell lysates were processed for coimmunoprecipitation with anti-CHK2, followed by probing with anti-FoxO3a, FoxK and FoxO4. IP, immunoprecipitation. (B) GCs transfected with CHK2 siRNA or scrambled control siRNA for 24 h/48 h were cultured for 24 h and then the cells were rinsed with PBS. The expression of CHK2 was determined by qPCR and the expression of CHK2 protein was determined by Western blot. Bars with different superscripts are statistically different (p < 0.05). (C) GCs transfected with CHK2 siRNA for 24 h were cultured in media containing ACNU. 24 h later, the cells were rinsed with PBS and the expressions of p53, p-p53, Rad51, CDKN1A, CCND1, LC3B and Atg7 were determined by Western blot. Bars with different superscripts are statistically different (p < 0.05).

Fig 5: The proposed mechanism of action of CB in ICC cells. CB treatment induces DNA damage and activates ATM and AKT. Activated ATM increases p-CHK2-T68 and p-p53-S15, which promotes the expression of FAS, DR4 and DR5 and triggers cell apoptosis. Activated AKT, meanwhile, promotes cell survival by activating mTOR and its downstream signaling pathway. However, the tumor cells underwent apoptosis after CB treatment due to the predominant activation of the ATM/CHK2/p53 apoptotic pathway.