Fig 2: M. avium induces IFN-ß production in macrophages through a cytosolic RIG-I/MAVS/TBK1/IRF3/7-depdendent pathway.(A) ELISA analysis for IFN-ß production in WT BMMs infected with M. avium 104 and 2151 at 24 and 72 hr post-infection. (B) qRT-PCR analysis for IFN-ß production in WT BMMs infected with M. avium 104 at various time points post-infection. The IFN-ß mRNA levels were expressed as fold change relative to M. avium-infected BMMs at 2 hr post-infection. (C) qRT-PCR analysis for mycobacterial RNA abundancy in M. avium-infected BMMs (Total Cellular RNA) or the cytosol (Cytosolic RNA) at 24 hr post-infection. Each M. avium 104 transcript abundance was expressed as the percentage relative to M. avium-infected BMMs (Total Cellular RNA). (D) qRT-PCR analysis for IFN-ß production in M. avium-infected WT and Mavs-/- BMMs at 24 hr post-infection. (E) Similar to (D), but IFN-ß protein was quantified by ELISA. (F) Similar to (D), but M.avium-induced IFN-ß production was determined in BMMs at 24 hr post-infection following a 48 hr pretreated with negative control (Control), RIG-I, TBK1, IRF3 or IRF7 siRNA. (G) Similar to (F), but M.avium-induced IFN-ß production was measured by ELISA. (H) Western Blot analysis for IRF3 and IRF7 in whole cell lysate (WCL) and nuclear fraction (Nucleus) of WT and Mavs-/- BMMs at 24 hr post-infection. (I) Similar to (H), but TBK1 phosphorylation (Ser172) was analyzed in WCL. ß-Actin and Histone H3 served as loading control for WCL and nuclear fraction, respectively. Data shown in (A-G) are the mean ±SD (n = 3 wells per group), and all data are representative of three independent experiments (biological replicates). For (D) and (F), the data was expressed as fold change relative to M.avium-infected WT (D) or control (F) BMMs. GAPDH served as loading control in qRT-PCR. *P < 0.05, **P < 0.01, and ***P < 0.001 by Student’s t-test (two-tailed).

Fig 3: FIP200 activates innate immunity via RIG-I but independently of its autophagy function.a FIP200 wild type and knockout HEK293 cells were transfected with vector, RIG-I, TBK1, MAVS, or IRF3(5D) together with pRL-SV40 and pISRE-Luc. After 48 h, cells were collected and the ratio of firefly luciferase to Renilla luciferase was calculated to determine the relative activity of IFN reporter. All experiments were biologically repeated three times. Data represent means ± s.d. of three independent experiments. The P-value was calculated (two-tailed Student’s t test) by comparison with HEK293 cells. *P < 0.05. b Wild-type HEK293 cells, FIP200 knockout cells reconstituted with full-length FIP200, delATG, or delClaw were stimulated with 1 µg ml-1 poly(I:C) for designated times. Then, RNA was extracted, and real-time PCR for IFNß was performed. All experiments were biologically repeated three times. Data represent means ± s.d. of three independent experiments. (**P < 0.01 vs. the wild-type cells by two-tailed Student’s t test). Right panel shows the expression of FIP200 and the mutants in the reconstituted cells. The anti-FIP200 antibody detects the C-terminal end of FIP200 and cannot recognize delATG and delClaw. c RIG-I was transfected with vector, FIP200, FIP200-4A, or ULK1 into HEK293 cells. After 48 h, RNA was extracted, and real-time PCR assays for IFNß, IP10, and RANTES were performed. All experiments were biologically repeated three times. Data represent means ± s.d. of three independent experiments. (*P < 0.05, **P < 0.01, by two-tailed Student’s t test). d Wild-type HEK293 cells, FIP200 knockout cells, and FIP200 knockout cells reconstituted with full-length FIP200 or the 4A mutant were stimulated with 1 µg ml-1 poly(I:C) for designated times. Then, RNA was extracted, and real-time PCR for IFNß was performed. All experiments were biologically repeated three times. Data represent means ± s.d. of three independent experiments. (*P < 0.05 vs. the wild-type cells by two-tailed Student’s t test). Right panel shows the expression of FIP200 and the mutant in the reconstituted cells.