Figure 3

Figure 3 Portimine-inhibited translation promotes ZAKα-dependent P38 activation and hNLRP1 inflammasome activation in epithelial cells.

(A) Determination of protein synthesis in HEK293 cells expressing or not NLRP1 in response to Sample II (1/20,000 dilution), Portimine A (4 ng/mL), Portimine B (400 ng/mL), or Pinnatoxins-H/G (40 ng/mL) by measuring puromycin incorporation after 2 h exposure. Immunoblots show lysates from one experiment performed at least three times. (B) Schematic representation of the mechanism of ZAKα/P38 stress kinases activation upon induction of Ribotoxic Stress Response (RSR). Phosphotag blotting of phosphorylated ZAKα in pHEK cells exposed to Sample II (1/20,000 dilution), Portimine A (4 ng/mL), Portimine B (400 ng/mL), or the known RSR inducer Anisomycin (1 µg/mL) for 8 h. When specified, PLX420 (b-Raf, ZAKα inhibitor, 10 µM) was used. Immunoblots show lysates from one experiment performed at least three times. (C) Phosphotag blotting of phosphorylated ZAKα, P38 and NLRP1 disordered Region (DR) in NTERT NLRP1 KO + 86-275-SNAP (described in Fig. EV3D) cells exposed to Portimine A (20 or 80 ng/mL) or to the known RSR inducer Anisomycin (1 µg/mL) for an hour. When specified, 6p (ZAKα inhibitor, 1 µM) was used. Ponceau staining and GAPDH were used as internal protein loading controls. Immunoblots show lysates from one experiment performed at least two times. (D) Immunoblotting of P38, ZAKα, Tubulin, and phosphorylated P38, plasma membrane permeabilization (SYTOX Green incorporation, 6 h) and IL-1β release evaluation (24 h) in pHEKs WT or genetically invalidated (CRISPR-Cas9) for ZAKα 8 h after exposure to Portimine A (4 ng/mL) or the known RSR inducer Anisomycin (1 µg/mL). ***P ≤ 0.0001, one-way ANOVA. Values are expressed as mean ± SEM. Graphs show one experiment performed in triplicates at least three times. (E) Fluorescence microscopy and associated quantifications of ASC-GFP specks in A549^ASC-GFP reporter cells expressing or not NLRP1 exposed to 4 ng/mL of Portimine A or to 1 µg/mL of Anisomycin for 6 h. When specified, SB 203580 (P38α/β inhibitor, 10 µM) was used. ASC-GFP (green) pictures were directly taken in dish after adding Hoechst (nuclei staining). Images shown are from one experiment and are representative of three independent experiments; scale bars, 10 µm. ASC complex percentage was performed by determining the ratios of cells positive for ASC speckles on the total nuclei (Hoechst). At least ten fields from each experiment were analyzed. Values are expressed as mean ± SEM. ***P ≤ 0.0001, one-way ANOVA. (F) Western blot showing NLRP1 using an anti-NLRP1 N-terminal antibody (aa 1–323) in HEK293^ASC-GFP reporter cells reconstituted with hNLRP1 or hNLRP1 plasmid constructs mutated (S107F and DelP108_T112) after 6 h exposure to Portimine A (4 ng/mL) or after 10 h exposure to Val-boro-Pro (VbP, 10 µM). Images shown are from one experiment and are representative of three independent experiments. (G) Fluorescence micrographs and respective quantifications of ASC-GFP specks in HEK293^ASC-GFP reporter cells reconstituted with hNLRP1 or hNLRP1 plasmid constructs mutated for S107F and DelP108_T112 after 6 h exposure to Portimine A (4 ng/mL) or after 10 h exposure to Val-boro-Pro (VbP, 10 µM). ASC-GFP (green) pictures were taken in the dish after toxin exposure. Images shown are from one experiment and are representative of three independent experiments; scale bars, 10 µm. ASC complex percentage was performed by determining the ratios of cells positive for ASC speckles (green, GFP) on the total nuclei (Hoechst). At least ten fields from three independent experiments were analyzed. Values are expressed as mean ± SEM. ***P ≤ 0.0001, two-way ANOVA with multiple comparisons. Graphs show one experiment performed in triplicate at least three times. Source data are available online for this figure.