Fig. 4: Cytokine production during necroptosis is mediated by a cell intrinsic pathway.
From: Necroptosis promotes cell-autonomous activation of proinflammatory cytokine gene expression
a The cell-intrinsic and paracrine signaling hypothesis. b Schematic of the treatments for HT-29 cells with indicated compound(s) or conditioned medium. N, 10 μM Nec-1s. c HT-29 cells were treated as shown in b. The expression of Cxcl8 was determined by qPCR; c.m., conditioned medium. d, e HT-29 cells expressing MLKL shRNA or p65 shRNA were cultured individually or co-cultured in a ratio 1:1, and stimulated with DMSO or TSZ. The mRNA levels of Cxcl8 and Cxcl1 were measured by qPCR after 8 h of treatment and the cell viability was determined by CellTiter-Glo after 29 h of treatment (d). The protein levels of p65 and MLKL were analyzed by western blotting (e). f Schematic of the co-culture and FACS experiment. HT-29 cells were treated with DMSO or TSZ for 6 h, and then separated by FACS to obtain Cherry+ (shControl) or GFP+ (shMLKL) populations. g Western blotting analysis of lysates from HT-29 cells separated in f. Asterisk indicates nonspecific bands. h Quantitative PCR analysis of Cxcl8, Cxcl1, Cxcl2, and Csf1 mRNA levels in HT-29 cells derived from f. i The expression of the cytokines was determined by RT-PCR in the attached or detached HT-29 cells after 8 h treatment with TSZ
