Fig. 9: IL-6-driven FasL in myeloid cells facilitated PUMA-mediated epithelial apoptosis in vitro.

a Schematic diagram of the co-culture experiments with primary myeloid cells and epithelial cells isolated from mice. b The concentration of FasL in the indicated medium of (a) was detected with ELISA (left panel, n = 6 per group). Values are presented as mean ± SEM. *P < 0.05 versus the medium of PBS-treated cells, ^#P < 0.05 versus the medium of IL-6-treated p65^f/f/PUMA-WT cells. Expressions of the related proteins of primary myeloid cells were detected by western blotting (right panel, n = 6 per group). c The extent of primary epithelial cells apoptosis from the co-culture experiments was preformed using an Annexin V-PE/7-AAD staining kit (Flow cytometric analysis). d The indicated proteins from primary epithelial cells in co-culture experiments were presented, and deletion of NF-κBp65 in myeloid cells of PUMA-WT mice repressed Fas, NF-κBp-p65, PUMA and cleaved caspase-3, without affecting Bid and caspase-8 cleavage, in epithelial cells under IL-6 treatment in co-culture (upper panel). The epithelial cells viability was analyzed by CCK-8 assay (lower panel). *P < 0.05 versus the medium of PBS-treated cells. e Western blotting depicted IL-6 treatment did not influenced the levels of Fas, FasL, PUMA and cleaved caspase-3 of the isolated primary epithelial cells. β-actin was used as the loading control. n = 6 per group. f FasL treatment enhanced the expressions of Fas, NF-κBp-p65, PUMA and cleaved caspase-3 of the isolated primary epithelial cells. β-actin was used as the loading control. n = 6 per group. g Mitochondrial and cytosolic fractions were analyzed for cytochrome c by western blotting (n = 6 per group). β-actin and COX IV were used as the loading control of cytosolic and mitochondrial fractions, respectively