Fig. 6. Host epithelial cell nitrate metabolism boosts C. concisus growth in vitro.

a,b, Growth of C. concisus after 8 a, or 24 hours b, in CT26 mouse colonic epithelial cell line supernatants that were live (green; CT26:FADD, z-VAD, z-VAD+BB) or were treated with an inducer of apoptosis (red; BB) compared to medium control (n=4 biological replicates per condition). c-e, Growth of C. concisus after 6 c,, 12 d,, or 24 e, hours in HTC116 human colonic epithelial cell line supernatants harvested from cells that were live (green; QVD, QVD+STS), or that had been treated with an inducer of apoptosis (STS; red), compared to cell culture medium alone control (n=3 biological replicates per condition). f, Expression of C. concisus nitrate reductase napA at 9 days post-isolate gavage, or in cSI-I and cSI-N sham-gavaged controls. g, Levels of nitrate in homogenized ileal tissue 9 days post-isolate gavage. h, Levels of nitrite in cecal contents 9 days post-isolate gavage. For (F-H), n=5–11 mice/group combined across two independent experiments. i,j, Growth of C. concisus after 8 (i) or 24 (j) hours of incubation in HTC116 human colonic epithelial cell line supernatants prepared from live cells (green; QVD, QVD+STS) or cells that had been treated with an inducer of apoptosis (red; STS). All cells were additionally treated with an inhibitor of nitric oxide synthase (L-NIO). Growth shown relative to cell culture medium alone (n=4 biological replicates per condition). k, Comparison of C. concisus growth after 24 hours in live (green; QVD, QVD+STS) or apoptotic (red; STS) HTC116 supernatants that were or were not treated with L-NIO (n=3–4 biological replicates per condition, pairwise t-tests for each ± L-NIO comparison). For all panels, bars denote mean ± s.d.. For a-e and g-k, P-values were determined with one-way ANOVA and Tukey’s multiple comparisons. FADD, Fas-associated death domain; z-VAD, z-VAD-FMK (pan-caspase inhibitor); QVD, Quinoline-Val-Asp-Difluorophenoxymethylketone (caspase inhibitor); STS, Staurosporine (apoptosis inducer); L-NIO, N⁵-(1-Iminoethyl)-L-ornithine, dihydrochloride (nitric oxide synthase inhibitor).