Extended Data Fig. 5: Generation of recombinant API5 protein and effect of environmental triggers on the secretome of IELs.
From: The γδ IEL effector API5 masks genetic susceptibility to Paneth cell death
a, Architecture of API5. The N and C termini, the HEAT and ARM-like domains, residues mutated in our study (Y8, Y11) are indicated. b, Size-exclusion chromatograms of Superdex200 (GE Healthcare) of wild-type and (Y8K:Y11K) rAPI5, indicating that they are predominantly monomeric. c, Thermal denaturation of API5 monitored using SYPRO Orange binding. The graph shows the first derivative of fluorescence intensity (n = 3). d, Viability of small intestinal organoids from Atg16L1ΔIEC (ΔIEC) mice cultured ± 50 nM and 200 nM wild-type or mutant (Y8K:Y11K) recombinant API5 for 48 h. e, Western blot analysis of SN samples harvested from TCRγδ+ IELs stimulated with anti-CD3/CD28 for 24 h corresponding to Fig. 2i. Total SN was equally divided into three; 1 left untreated (Pre-IP), and the other 2 were immunoprecipitated with anti-API5 antibody or control IgG antibody-conjugated magnetic beads for 24 h. f, g, Representative images (f) and viability (g) of small intestinal organoids from ΔIEC mice stimulated ± 0.5 ng/ml IFNγ for 48 h after pretreatment with 50 nM wild-type rAPI5 for 3 days. Scale bar 25 μm. h, i, Representative H&E images (h) and quantification (i) of Paneth cell and total IEC number per organoid stimulated ± 0.5 ng/ml IFNγ ± 50 nM wild-type rAPI5 for 24 h after pretreatment with 50 nM wild-type rAPI5 for 4 days. Arrowheads indicate Paneth cells. Scale bar 20 μm. j, Quantification of the indicated cytokines in SN of γδ IELs harvested either from naïve or MNV-infected WT mice (n = 5 per condition). k, Western blot analysis of SN from γδ IELs harvested from naïve, Salmonella-infected, or indomethacin-treated WT mice following stimulation with anti-CD3/CD28 for 24 h. l, Quantification of API5 normalized to PGRP-L by densitometric analyses of (k). Each value is divided by naïve. In organoid experiments, intestinal crypts were harvested from 3 mice per genotype. Two-sided unpaired Student’s t-test in (d, j). An ANOVA with Tukey’s multiple-comparison test in (g, i). Two-sided paired Student’s t-test in (l). Data points in (d) and (g) represent organoid viability in each well, data points in (i) represent individual organoids, data points in (j) represent individual mice, and data points in (l) represent API5/PGRP-L value in each western blot. Bars represent means ± SEM, and at least two independent experiments were performed.
