1. Volume 19, Issue 7, Pages 1418-1430 (May 2017)
Innate Recognition of Intracellular Bacterial Growth Is Driven by the TIFA-Dependent Cytosolic Surveillance Pathway 
Ryan G. Gaudet, Cynthia X. Guo, Raphael Molinaro, Haila Kottwitz, John R. Rohde, Anne-Sophie Dangeard, Cécile Arrieumerlou, Stephen E. Girardin, Scott D. Gray-Owen 
Cell Reports 
Volume 19, Issue 7, Pages (May 2017)
DOI: /j.celrep
Copyright © 2017 The Author(s) Terms and Conditions

2. Cell Reports 2017 19, 1418-1430DOI: (10.1016/j.celrep.2017.04.063)
Copyright © 2017 The Author(s) Terms and Conditions

3. Figure 1
TIFA Mediates NOD1- and NOD2-Independent Detection of Invasive Shigella
(A) ELISA of IL-8 in the supernatants of wild-type (WT) NOD1, NOD2 knockout (KO), or NOD1 and NOD2 double KO (dKO) HCT cells infected with invasive Shigella flexneri M90T (S.f) or non-invasive BS176, or treated as indicated for 6 hr.
(B and C) ELISA of IL-8 at 6 hr (B) or IL8 mRNA by qPCR (C) in TIFA or NOD1 KO cells expressing NOD1- or TIFA-targeting shRNAs, then infected with S.f. Inset: immunoblot indicates endogenous TIFA protein expression.
(D and E) Immunoblot of phosphorylated (p) IκBα (top) or TAK1 (bottom) (D), or nuclear localization of NF-κB p65 by microscopy (E) in WT or TIFA KO HCT cells infected with S.f. Blots for p-TAK1 and β-actin were exposed equally for both genotypes. Scale bars, 10 μm.
(F and G) NF-κB luciferase activity (F) or IL-8 by ELISA (G) in WT or TIFA KO 293T cells infected with S.f (MOI = 10), or stimulated with either soluble lysate or cell-free supernatant (CFS) derived from 100-fold the amount of bacteria used in the infection.
(H) ELISA of IL-8 or CFU recovered from HCT cells infected with S.f (6 hr) of the indicated genotype. ADP-heptose biosynthetic pathway of enteric bacteria is indicated on the left. Unless otherwise indicated, values represent mean ± SEM. Results represent three independent experiments or are representative of two or three independent experiments (D and E). ∗p < 0.05, ∗∗∗p < by ANOVA.
See also Figures S1–S4.
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4. Figure 2
TIFA Responds to Gram-Negative Bacteria that Escape the Vacuole
(A and B) NF-κB luciferase activity in wild-type or TIFA KO 293T cells infected with the indicated bacteria (A) or stimulated with lysates derived from S.f or Salmonella typhimurium in the presence or absence of digitonin (Dig) (B).
(C) TIFA aggregation measured by clear-native PAGE (CN-PAGE) in 1XFLAG-TIFA expressing 293T cells infected with WT or ΔsifA Salmonella for 6 hr.
(D and E) shRNA knock down of MyD88 and NF-κB luciferase in WT or TIFA KO 293T cells after 8 hr (D) or ELISA of IL-8 production by WT or TIFA KO HCT cells after 20 hr (E) of infection with Salmonella of the indicated genotype.
(F) NF-κB luciferase activity in WT or TIFA KO 293T infected with Listeria monocytogenes (L.m) for 6 hr in the presence of increasing amounts of HBP.
(G) IFNB1 mRNA induction by qPCR in HCT cells infected with L.m in the presence or absence of HBP. Data represent greater than or equal to three independent experiments. ns, not significant (p > 0.05). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < by ANOVA.
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5. Figure 3 NOD1 and TIFA Sequentially Respond to Invasive Shigella
(A–C) Time course of p65 nuclear translocation by transcription factor ELISA (A) or immunoblot analysis of phosphorylated (p)-IκBα (B) or SAPK/JNK (C) in HCT cells of the indicated genotype during infection with S.f. All blots in (C) were exposed for the same amount of time.
(D–F) Time course of IL8 mRNA by qPCR (D), or IL-8 protein by ELISA (6 hr) (E and F) during infection of HCT cells with S.f (MOI = 50) or at the indicated dose (F).
(G) HCT cell death measured by 7-AAD staining 6 hr after infection with S.f (MOI = 50).
Results represent greater than or equal to three independent experiments or are representative of greater than or equal to two independent experiments (B and C). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < by ANOVA.
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6. Figure 4
TIFA Expression Dictates the Cellular Responsiveness to HBP and Sensitivity to Invasive Shigella
(A) TIFA and NOD1 expression by qPCR in primary human tissues pooled from healthy donors (n ≥ 5).
(B–E) TIFA mRNA by qPCR in HCT cells (B), in 293T cells overexpressing NOD1 in the presence or absence of C12-iE-DAP (C), in HCT cells deficient in RelA (D), or infected with S.f for 6 hr (E). The inset in (D) shows protein expression in shRNA-treated cells.
(F) NF-κB luciferase activity in WT or TIFA KO 293T cells expressing TIFA in trans, primed as indicated, then infected with S.f.
(G and H) IL8 expression by qPCR in HCT cells treated with cycloheximide (CHX) and infected for 3 hr with S.f at the indicated dose (G) or at MOI = 50 for the indicated time (H).
(I) Relative gene expression by qPCR in the indicated human tissues.
(J) IL8 mRNA by qPCR or protein by ELISA following transduction of Caco-2 cells with an empty or TIFA-encoding retrovirus and treated as indicated.
(K) Tifa expression by qPCR in pooled primary murine tissues (n = 3) compared with the mIC-Cl2 cell line.
(L and M) KC and MIP-2 levels by ELISA (L) or qPCR (M) in mIC-CI2 cells expressing TIFA in trans and treated as indicated (M). Values (A and K) and are mean ± SD of three technical replicates. All other values represent greater than or equal to three independent experiments. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < by ANOVA.
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7. Figure 5 Shigella Induces TIFA Aggregation and TRAF6 Activation
(A–D) Cell-fractionation and immunoblot (IB) for: FLAG-TIFA in 293T cells (A and B), FLAG-TIFA in TIFA KO cells complemented with the indicated allele of 1XFLAG-TIFA (C), or TRAF6 and lysine 63 (K63)-linked ubiquitin (Ub) in wild-type or TIFA KO 293T cells infected with S.f for 4 hr (D). Detergent soluble or insoluble (pellet) fractions were separated under native or denaturing (denat) conditions.
(E and F) Immunoprecipitation (IP) and IB analysis (4 hr) (E) or ELISA measurement of IL-8 in the supernatants (6 hr) (F) of S.f-infected TIFA KO 293T cells complemented with the indicated allele of 1XFLAG-TIFA.
(G) TIFA aggregation in S.f-infected HCT WT or NOD1 KO cells expressing 1XFLAG-TIFA. Scale bars, 10 μm.
(H) Time course of TIFA aggregation in S.f (red)-infected 293T TIFA KO cells complemented with 1XFLAG-TIFA (green). Results are representative of greater than or equal to three independent experiments. Scale bars, 5 μm. Quantification represent mean ± SEM of ≥20 fields in each of three independent experiments. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < by ANOVA.
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8. Figure 6
TIFA Responds to HBP Released by Gram-Negative Bacteria during Growth
(A) Assay used to assess TIFA-stimulating activity released from S.f.
(B and C) ELISA of IL-8 production by HCT cells in the presence or absence of digitonin (Dig) stimulated with cell-free supernatants (CFS) from wild-type (B) or mutant (C) S.f or E. coli.
(D) NF-κB luciferase activity from permeabilized WT or TIFA KO 293T cells overexpressing NOD1 (pNOD1) and stimulated with E. coli CFS.
(E) CFS from S.f grown at 12°C or 37°C presented into permeabilized FLAG-TIFA 293T cells and analyzed by clear-native (CN) PAGE.
(F) S.f grown at different densities (x axis) in cytoplasm extracts for 2 hr and growth rate determined (right y axis). CFS was harvested, normalized to final CFU/ml, presented to permeabilized TIFA- or NOD1-KO HCT cells and IL-8 measured by ELISA (left y axis).
(G) IL-8 production, described in (F), plotted versus the fold increase in S.f from which the CFS was derived.
(H) TIFA oligomerization by CN PAGE in 293T cells stimulated with the CFS from normalized S.f cultures starting from the indicated growth stage (m = mock).
(I) ELISA of IL-8 produced by permeabilized primary human colonocytes treated with CFS derived from S.f grown for 2 hr in tryptic soy broth (TSB). Results represent two (E, I, and H) or three (A–D, F, and G) independent experiments.
See also Figures S5 and S6.
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9. Figure 7
Invasive but Metabolically Attenuated Shigella Fail to Activate TIFA
(A–D) Quantification of S.f of the indicated genotype recovered after infecting HCT cells (A and D), and ELISA measurement of IL-8 in the supernatants 6 hr (B) or 1 hr (C) after infection.
(E) IL8 mRNA by qPCR at the indicated time in HCT WT or TIFA KO cells infected with S.f.
(F and G) Recovered S.f of the indicated genotype (F) or ELISA measurement of IL-8 produced 6 hr after infection (G) in the presence or absence of 100 mM acetyl-phosphate.
(H) NF-κB luciferase activity in 293T cells of the indicated genotype, transfected with pTIFA or pNOD1, and infected with the indicated genotype of S.f for 6 hr in the presence or absence of acetyl-phosphate. Results represent three independent experiments. ns, not significant. ∗∗p < 0.01, ∗∗∗p < by ANOVA.
Cell Reports  , DOI: ( /j.celrep )
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