Fig 2: NLRP6 interferes with enterocyte iron metabolism upon S. Typhimurium infection. (A–F) Wild-type and NLRP6-/- Caco-2 cells were infected with S. Typhimurium strain SL1344 at an MOI of 50 for the indicated time points. (A) Non-heme iron content in Caco-2 cells (n = 4 independent experiments). (B) Western blot analysis and relative intensity of FPN (n = 4 independent experiments). (C) Quantitative PCR analysis of FPN (n = 4 independent experiments). (D) Western blot analysis and relative intensity of KEAP1 and NRF2 (n = 4 independent experiments). (E) Quantitative PCR analysis of NQO1 (n = 4 independent experiments). (F) Western blot analysis and relative intensity of p-AKT (Ser473) and AKT (n = 4 independent experiments). (G, H) Wild-type and NLRP6-/- Caco-2 cells were treated with either vehicle or 1 µM MK2206 for 2 h and then infected with S. Typhimurium strain SL1344 at an MOI of 50 for 16 h. (G) Non-heme iron content in Caco-2 cells (n = 4 independent experiments). (H) Quantitative PCR analysis of NQO1 and FPN (n = 4 independent experiments). (I, J) Wild-type and NLRP6-/- Caco-2 cells were infected with S. Typhimurium strain SL1344 at an MOI of 50 for the indicated time points. (I) Western blot analysis and relative intensity of LC3 (n = 4 independent experiments). (J) Western blot analysis and relative intensity of NBR1, TAX1BP1, OPTN, p62 and NDP52 (n = 4 independent experiments). Data were compared with independent Student's t-test. Values are expressed as the mean ± SEM, and statistically significant differences are indicated. *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant.

Fig 3: TAX1BP1 was the major receptor of CuONPs-induced mitophagy in vascular endothelial cells. A MTS analysis was performed to detect EA.hy926 cell viability. Cells were transfected with siNC, siGFP, siTAX1BP1, siNBR1, sip62, siOPTN or siNDP52 for 48 h, and then treated with CuONPs (0, 5, 7.5, 10 and 15 µg/ml) for 24 h. B Representative immunofluorescence images indicates colocalization of TAX1BP1 with GFP-LC3 and Mito-DsRed dots in EA.hy926 stable cell line expressing GFP-LC3 and Mito-DsRed after treatment with 10 µg/ml CuONPs for 12 h. White arrow indicate TAX1BP1-positive dots that colocalized with GFP-LC3 and Mito-DsRed signals. Scale bars, 20 µm. C and D Representative FCM results of EA.hy926 cells labeled with MitoTracker Green FM (C) and MitoSOX (D), respectively. The cells were transfected with siNC or siTAX1BP1 for 48 h and then treated with CuONPs (10 µg/ml) for 12 h. All data were statistically analyzed using one-way ANOVA with Tukey’s test. Results are representative of at least three independent experiments. Data are mean ± S.D. *p < 0.05

Fig 4: TBK1 aggregates are positive for the autophagy receptors TAX1BP1 and SQSTM1/p62 pS403. (A + B) fip200 KO MEFs transfected with an empty vector (A) or cDNA encoding FIP200 ?CT (B) were fixed in 100% MeOH and immunostained for TBK1 in combination with TBK1 pS172, FIP200, SQSTM1/p62 pS403, NDP52, or OPTN. Representative sections of three independent experiments are depicted. The bar graphs represent the pixel intensities of the areas indicated by the respective dashed red arrows shown in the insets.

Fig 5: Loss of Nlrp6 impairs autophagy during S. Typhimurium infection. (A) HeLa cells were transfected with GFP-RFP-LC3 together with either an empty vector or His-NLRP6. After 36 h of incubation, HeLa cells were infected with S. Typhimurium strain SL1344 at an MOI of 10 and analyzed at 6 h post-infection. Fluorescence microscopic analysis of LC3. (Uninfected, 18 cells per group; Infected, 24 cells per group; n = 3 independent experiments). Original magnification, × 100; scale bars, 10 µm. Representative fluorescence images are shown. The number of LC3 puncta was counted using ImageJ Launcher broken symmetry software program. (B, D–F) BMDMs isolated from wild-type and Nlrp6-/- mice were infected with S. Typhimurium strain SL1344 at an MOI of 10 for the indicated time points. (B) Western blot analysis and relative intensity of LC3 (n = 4 independent experiments). (C) BMDMs isolated from wild-type and Nlrp6-/- mice were treated with either vehicle or 200 nM BafA1 for 4 h and then infected with S. Typhimurium strain SL1344 at an MOI of 10. Western blot analysis and relative intensity of LC3 at 4 h post-infection (n = 4 independent experiments). (D) Quantitative PCR analysis of Lc3 (n = 4 independent experiments). (E) Quantitative PCR analysis of Lamp1 (n = 4 independent experiments). (F) Western blot analysis and relative intensity of NBR1, TAX1BP1, OPTN, p62 and NDP52 (n = 4 independent experiments). Data were compared with independent Student's t-test. Values are expressed as the mean ± SEM, and statistically significant differences are indicated. *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant.