Fig 2: NLRP1 activation is mediated by its ubiquitination. Protein expression levels of NLRP1 (a) in HaCaT cells pre-treated or not with the proteasome inhibitor MG-132 and then exposed to O3. (b) Double immunofluorescence staining for NLRP1 (green) and ASC (red), in HaCaT cells pre-treated with MG-132 and then exposed to O3. Blue staining (DAPI) represents nuclei. Images were taken at 40 × magnification (scale bar = 20 μm). The fluorescent levels were quantified using ImageJ software. Protein expression levels of p20 Caspase 1 over Pro-Caspase 1 (c), and UBR2 (d) in HaCaT cells pre-treated with MG-132 and exposed to O3. (e) Double IF staining for NLRP1 (red) and UBR2 (green) in HaCaT cells pre-treated with MG-132 and exposed to O3. Blue staining (DAPI) represents nuclei. Images were taken at 40 × magnification (scale bar = 20 μm). For all the experiments HaCaT cells were pre-treated or not with the proteasome inhibitor MG-132 20 μM for 2 h and then exposed to O3 0.4 ppm for 1 h. Samples were collected at the indicated timepoints post 1-h O3 exposure. For all western blotting the protein expression level was quantified using ImageJ software and β-actin or Red ponceau were used as internal control. Data are the results of the averages of at least three different experiments, *p < 0.05 and #p < 0.05 by 2-way ANOVA followed by Tukey’s post-hoc comparison test. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig 3: O3activated NLRP1 inflammasome via Caspase I (a) Immunofluorescence staining for NLRP1 (green) and ASC (red) in HaCaT cells silenced for NLRP1 10 nM for 24 h and exposed to O3. The blue staining (DAPI) represents nuclei. Images were taken at 40 × magnification (scale bar = 40 μm) and the fluorescent signal was quantified using ImageJ software. (b) Protein expression levels and relative quantification graph of p20 Caspase 1 over pro-Caspase 1 in HaCaT cells silenced for NLRP1 10 nM and then exposed to O3. (c) IL-1β released levels in media of HaCaT cells silenced for NLRP1 10 nM for 24 h and then exposed to O3. (d) Protein expression levels of p20 Caspase 1 over pro-Caspase 1 in HaCaT cells pre-treated with Caspase 1 inhibitor Z-YVAD-fmk 2 and 10 μM for 1 h and then exposed to O3. IL-1β mRNA expression levels (e) and released levels of IL-1β (f) in HaCaT cells pre-treated with Caspase 1 inhibitor Z-YVAD-fmk 2 μM for 1 h and then exposed to O3. For all the experiments HaCaT cells were exposed to 0.4 ppm O3 for 1 h and then collected at the indicated timepoints post 1-h O3 exposure. For all western blotting the protein expression level was quantified using ImageJ software and β-actin was used as internal control. Data are the results of the averages of at least three different experiments, *p < 0.05 and #p < 0.05 by 2-way ANOVA followed by Tukey’s post-hoc comparison test. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig 4: (A) Immunocytochemical staining images for NALP1 protein (red staining) and nuclei staining (DAPI) in HaCaT cells at 0, 3, and 6 h (T0, T3, and T6) after 15 min of exposure to CS. Images were taken at 40 × magnification, scale bar = 20 µm; immunofluorescence signal was quantified using ImageJ software (bottom panel). (B) Immunocytochemical staining for the co-localization of ASC protein (green) and NALP1 (red staining) in HaCaT cells 0, 3, 6, and 24 h (T0, T3, T6, and T24) after 15 min of CS exposure or unexposed cells. Blue staining (DAPI) represents nuclei; images were taken at 40 × magnification; scale bar = 20 µm. The NLRP1-ASC expression levels were measured by using ImageJ software (bottom panel). (C) Levels of released IL-1ß (upper panel) and IL-18 (bottom panel) (pg/mL) in media of HaCaT cells 24 h post 15 min of CS exposure. (D) Transcription levels of IL-1ß and IL-18 measured by qRT-PCR in HaCaT samples exposed for 15 min to CS and collected at 0, 3, 6, 12, 18, and 24 h (T0, T3, T6, T12, T18, and T24) upon the end of the 15-min CS exposure. RPL11 was used as reference gene. Data are the results of the averages of at least three different experiments, *p < 0.05 by one-way ANOVA

Fig 5: NLRP1 inflammasome activation by H2O2(a) H2O2 levels production in media of HaCaT cells pre-treated with 1000 U/ml of Catalase (Cat) for 2 h and then exposed to O3 assessed by AmplexRed assay. (b) mRNA expression levels of NLRP1 (upper panel) and ASC (bottom panel) in HaCaT cells pre-treated with 1000 U/ml of Cat for 2 h and then exposed to O3.(c) Double Immunofluorescence staining for NLRP1 (red) and ASC (green) in HaCaT cells pre-treated with Cat 1000 U/ml for 2 h and then exposed to O3. Blue staining (DAPI) represents nuclei. Images were taken at 100 × magnification (scale bar = 2.5 µm); the fluorescent levels were quantified using ImageJ software. (d) Caspase 1 released levels in media of HaCaT cells pre-treated with Cat 1000 U/ml for 2 h and then exposed to O3. (e) IL-1ß mRNA expression levels in HaCaT cells pre-treated with Cat 1000 U/ml for 2 h and then exposed to O3.(f) Released levels of IL-1ß in media of HaCaT cells pre-treated with Cat 1000 U/ml for 2 h and then exposed to O3. For all the experiments HaCaT cells were exposed to 0.4 ppm O3 for 1 h. Samples were collected at the indicated timepoints post 1-h O3 exposure. For all western blotting the protein expression level was quantified using ImageJ software and ß-actin was used as internal control. Data are the results of the averages of at least three different experiments, *p < 0.05 and #p < 0.05 by 2-way ANOVA followed by Tukey’s post-hoc comparison test. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)