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. Author manuscript; available in PMC: 2020 Feb 21.
Published in final edited form as: Cell. 2019 Feb 21;176(5):1014–1025.e12. doi: 10.1016/j.cell.2019.01.037

Figure 1. Microbiota-derived NO mediates widespread protein S-nitrosylation in C. elegans, including Argonaute proteins.

Figure 1.

(A) Robust S-nitrosylation of the C. elegans proteome by B. subtilis. Silver stain of endogenous SNO-proteins (following SNO-RAC) was performed on lysates harvested from C. elegans grown either on wild type B. subtilis 1A1 (WT) or Δnos B. subtilis (Δnos), treated with (SNO-proteome; left panel) or without (Control; middle panel) ascorbate (Asc). Coomassie blue stain of total proteome loading controls (right panel). Gels are representative of three experiments. (B) Quantification of gels in A (n=3, ± SEM). *, differs from WT by ANOVA with Dunnett’s test (p < 0.05). (C) S-nitrosylation of the C. elegans proteome by E. coli. Silver stain of endogenous SNO-proteins (following SNO-RAC) was performed on lysates harvested from C. elegans grown either on wild type E. coli(WT) or ΔnarG E. coli (ΔnarG), and either treated with (SNO-proteome; left panel) or without (Control; middle panel) ascorbate. Coomassie blue stain of total proteome loading controls (right panel). Gels are representative of three experiments. (D) Quantification of gels in C (n=3, ± SEM). *, differs from WT by ANOVA with Dunnett’s test (p < 0.05). (E) S-nitrosylation of C. elegans ALG-1 by NO derived from B. subtilis NOS. ALG-1 immunoblot following SNO-RAC (+Asc) from lysates as in A. -Asc serves as a control (Forrester et al., 2007). Total ALG-1 loading control is also shown. Gels are representative of three experiments. (F) Quantification of gels in E (n=3, ± SEM). *, differs from WT by ANOVA with Dunnett’s test (p < 0.05). (G) S-nitrosylation of C. elegans ALG-1 by NO derived from E. coli nitrate reductase NarG. ALG-1 immunoblot following SNO-RAC (+Asc) from lysates as in A. Total ALG-1 loading control is also shown. Gels are representative of three experiments. (H) Quantification of gels in G (n=3, ± SEM). *, differs from WT by ANOVA with Dunnett’s test (p < 0.05). (I) Robust S-nitrosylation of the C. elegans proteome by small amounts of B. subtilis: effect of titration. Coomassie blue stain of endogenous SNO-proteins (following SNO-RAC) was performed on lysates harvested from C. elegans grown either on wild type (WT) B. subtilis 1A1 (100%) or Δnos (Δnos) B. subtilis 1A1 (0%) or a mixture comprising 10% WT and 90% Δnos; the SNO-proteome, -Asc control and total proteome loading controls are shown (left to right, respectively). Gels are representative of three experiments. (J) Quantification of gels in I (n=3, ± SEM). *, differs from WT by ANOVA with Dunnett’s test (p < 0.05). (K) S-nitrosylation of C. elegans ALG-1 by small amounts of B. subtilis NOS. Immunoblot of endogenous ALG-1 (following SNO-RAC) was performed on lysates harvested from C. elegans grown on wild type (WT) B. subtilis 1A1 (100%) , Δnos (Δnos) B. subtilis 1A1 (0%), or mixtures comprising 10% WT and 90% Δnos, 25% WT and 75% Δnos, 50% WT and 50% Δnos, and 75% WT and 25% Δnos. A lower exposure autoradiography film is shown (middle). Total ALG-1 loading control is also shown. Gels are representative of three experiments. (L) Quantification of gels in J (n=3, ± SEM). *, differs from WT by ANOVA with Dunnett’s test (p < 0.05). (M) Bacterial colony forming units (CFU) per C. elegans from worms cultured with either WT B. subtilis (WT) or Δnos B. subtilis (Δnos). See also Tables S1 and S2.