Fig. 4: Caspase-3/-8 independent ATG16L1 protein degradation and induction of pro-inflammatory responses.

a THP-1 macrophages were stimulated with Eh for different time points to detect caspase-3 activation. b THP-1 macrophages were pre-treated with caspase-6 and caspase-3 inhibitor Z-VEID-fmk and Z-DEVD-fmk, respectively for 1 h with 50 μM concentration prior to stimulation with Eh for 10 min. ATG16L1 protein restoration was assessed by western blot along with lamin A/C restoration. c Bone marrow derived macrophages (BMDMs) were pre-treated with caspase-8 and caspase-3 inhibitor Z-IETD-fmk and Z-DEVD-fmk, respectively, for 1 h 50 μM concentration prior to stimulation with Eh for 10 min. ATG16L1 protein degradation was assessed by western blot along with ATG5 dissociation from ATG5-ATG12 conjugate and lamin A/C degradation. THP-1 macrophages were stimulated with Eh for 60 min in absence and presence of Z-VEID-fmk (50 μM) to detect d, TNF-α mRNA expression e, caspase-1 activation and IL-1β/IL-18 secretion. f Mouse proximal colon tissue from Eh (1 × 106) inoculated with 3 h closed colonic loops were quantified for ATG16L1 degradation by band densitometric measurement. g Pro-inflammatory cytokines and chemokines mRNA expression in corresponding colonic loop tissues after Eh inoculation. h Pro-inflammatory cytokines and chemokines secretion from BMDMs after Eh stimulation for 3 h. Cells without treatment is symbolized as (−ve). Eh-macrophage 1:20 ratio used for (a–e). Data are representative of at least three independent experiments (n = 3) and for statistical significance, t-test and one-way ANOVA followed by post hoc Bonferroni test was done. *P < 0.05, **P < 0.01, ***P < 0.001. Bars represent mean ± SEM.