Fig 2: NLRP6 interacts with AKT and regulates NRF2 activation via AKT-mediated phosphorylation of p62 and FOXO3A. (A, B) BMDMs isolated from wild-type and Nlrp6-/- mice were infected with S. Typhimurium strain SL1344 at an MOI of 10 and analyzed at 4 h post-infection. (A) Western blot analysis and relative intensity of p-p62 (Ser351) (n = 4 independent experiments). (B) Western blot analysis and relative intensity of p-4EBP1 (Thr37/46) and 4EBP1 (n = 4 independent experiments). (C, D) BMDMs isolated from wild-type and Nlrp6-/- mice were treated with either vehicle or 1 µM Rapamycin for 6 h and then infected with S. Typhimurium strain SL1344 at an MOI of 10 for 4 h. (C) Western blot analysis and relative intensity of p-4EBP1 (Thr37/46), 4EBP1 and p-p62 (Ser351) (n = 4 independent experiments). (D) Quantitative PCR analysis of Nqo1 (n = 4 independent experiments). (E, F) BMDMs isolated from wild-type and Nlrp6-/- mice were infected with S. Typhimurium strain SL1344 at an MOI of 10 and analyzed at 4 h post-infection. (E) Quantitative PCR analysis of Keap1 (n = 4 independent experiments). (F) Western blot analysis and relative intensity of KEAP1 (n = 4 independent experiments). (G) HEK293T cells were transfected with KEAP1 luciferase reporter plasmid together with either an empty vector or His-NLRP6. A plasmid expressing Renilla luciferase was co-transfected as an internal control. KEAP1 luciferase activity at 24 h post-transfection (n = 5 independent experiments). (H) HeLa cells transfected with either an empty vector or His-NLRP6 were infected with S. Typhimurium strain SL1344 at an MOI of 10 and analyzed at 6 h post-infection. Immunofluorescence microscopic analysis of the subcellular distribution of FOXO3A. Original magnification, × 40; scale bars, 50 µm. Representative fluorescence images are shown. (I, J) BMDMs isolated from wild-type and Nlrp6-/- mice were infected with S. Typhimurium strain SL1344 at an MOI of 10 and analyzed at 4 h post-infection. (I) Western blot analysis and relative intensity of p-FOXO3A (Ser253) and FOXO3A (n = 4 independent experiments). (J) Western blot analysis and relative intensity of p-AKT (Ser473) and AKT (n = 4 independent experiments). (K) HEK293T cells were transfected with Flag-AKT together with either an empty vector or GFP-NLRP6 (left), or transfected with combinations of GFP-NLRP6 and Flag-AKT or its empty vector (right) for 48 h. Coimmunoprecipitation of AKT and NLRP6. (L–N) BMDMs isolated from wild-type and Nlrp6-/- mice were treated with either vehicle or 1 µM MK2206 for 2 h and then infected with S. Typhimurium strain SL1344 at an MOI of 10 for 4 h. (L) Non-heme iron content in BMDMs (n = 4 independent experiments). (M) Quantitative PCR analysis of Fpn, Nqo1 and Keap1 (n = 4 independent experiments). (N) Western blot analysis and relative intensity of p-FOXO3A (Ser253), FOXO3A, p-p62 (Ser351), p-AKT (Ser473) and AKT (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; ns, not significant.

Fig 3: The changes in host iron metabolism and antimicrobial defense resulting from Nlrp6-deficiency are reversed by pharmacological inhibition of AKT in vivo. Streptomycin-pretreated wild-type and Nlrp6-/- mice were orally infected with 1 × 107 CFUs of S. Typhimurium strain SL1344. After 1 day of infection, mice were orally administrated with either vehicle or 120 mg/kg MK2206 and analyzed 3 days post-infection. (A) Viable count of S. Typhimurium in MLN, liver and spleen (n = 4–5 per group). (B) Serum iron levels (n = 4 per group). (C) Non-heme iron content in liver and spleen (n = 5 per group). (D) Non-heme iron content in isolated liver macrophages (n = 5 per group). (E) Western blot analysis and relative intensity of NRF2, FPN, p-AKT (Ser473), AKT, p-4EBP1 (Thr37/46), 4EBP1, p-FOXO3A (Ser253), FOXO3A and KEAP1 (n = 3 per group). (F) Model of NLRP6 in regulating host iron metabolism during S. Typhimurium infection. 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; ns, not significant.

Fig 4: PPARd knockdown abrogated the effects of GW501516 on 6-OHDA-triggered protein levels of DMT1, FPN1, and Ferritin, and mRNA expression of DMT1. Cells stably expressing shRNA targeting scrambled sequences or PPARd were pretreated with DMSO or GW501516 for 8 h, and subsequently incubated with or without 6-OHDA for 16 h. Total RNA and protein were extracted, and levels of mRNA and protein were analyzed by real-time PCR (A–E) and Western blot (F). RPS18 and a-tubulin were used as internal controls for real-time PCR and Western blot, respectively. Results are expressed as means of triplicate ± SE (A–D). * p < 0.05, ** p < 0.01 relative to untreated group; # p < 0.05 relative to 6-OHDA-treated group.

Fig 5: Comparison in western blotting for ferritin and TfR1 proteins between heated and unheated samples from Caco-2 cells treated with iron.Caco-2 cells were untreated or treated with 50uM hemin, 250 and 500uM FAC, or 25uM DFO for 24hr. 25ug of Caco-2 WCLs along with 10ug of WCL from HEK293 cells transfected with pCMV or pCVMFpn1 were heated at 95°C for 5min or not heated (at room temperature). They were subjected to successive western blotting with A) anti-Fpn1 (SLC40A1), B) anti-DMT1 (SLC11A2), C) anti-ferritin H, D) anti-TfR1, and E) anti-GAPDH antibodies. The larger area of Fpn1 western blot for HEK293 cells transfected with pCMV or pCVMFpn1 is shown right next to the panel A. The arrow indicates the transfected Fpn1 protein stuck on the top of the separation gel. The asterisk in D) may be a TfR1 dimer only seen in unheated samples. The experiments were repeated three times and the representative western blots are shown. The same experiment repeated in HepG2 cells are shown in S2 Fig.