Fig 2: P4 NF-CM induces pyroptosis(A) Phase-contrast images of FE25 cells treated with P4 NF-CM or DMSO NF-CM for 48 h.(B) Scanning electron microscopy (SEM) analysis of FE25 cells treated with P4 NF-CM for 0, 24, and 36 h.(C) Viability of FE25 cells 48 h after P4 NF-CM treatment with or without pretreatment of 5 mM MLKL blocker (NSA) or 5 mM PI3K inhibitor (3-MA). *p < 0.05 for the DMSO NF-CM-treated cells only.(D and E) Expressions of NLRP3, NLRP1, Caspase-1, RPK1 and p-PARP in P4 NF-CM-treated FE25 cells was assessed by western blot (WB). The expression and intracellular localization of Caspase1 (F), GSDMD and GSDMD-N terminal (G) were verified using confocal microscopy. Nuclei were stained by DAPI (blue).(H) Expression of GSDMD and GSDMD-N terminal in P4 NF-CM-treated FE25 cells with or without RU486 (PR inhibitor) was assessed by western blot (WB).(I) IHC showed GSDMD high-expressed in oviduct epithelium of P4 intrabursal-ID8-C57BL/6 mice.(J) ELISA measured the release of IL-1ß from NF-CM-treated FE25 cells. Vehicle treated with conditioned medium of NFs treated with 1‰ DMSO; NF-CM only, conditioned medium without any treatment; GSDMD-N, cleaved-GSDMD. *p < 0.05 compared with the control group (Student’s t test). The data represent mean ± SEM.