Journal: Redox biology

Article Title: Ubiquitination as a key regulatory mechanism for O 3 -induced cutaneous redox inflammasome activation.

doi: 10.1016/j.redox.2022.102440

Figure Lengend Snippet: Fig. 1. O3 activated 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 in hibitor 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.)

Article Snippet: Coverslips were incubated with primary antibody ASC (Cat. NBP1-78977 Novus Biological, Littleton, CO, USA) 1:100, NLRP1 (sc-166368 Santa Cruz Biotechnology Inc., Dallas, TX, USA) 1:50, 4HNE (ab46545, Abcam, Cambridge, UK) 1:400, DPP9 (ab42080, Abcam, Cambridge, UK)1:400, UBR2 (18852-1-AP, Proteintech Group Inc., Rosemont, IL, USA) 1: 150 in 0.25% BSA/PBS overnight at 4◦C.

Techniques: Immunofluorescence, Staining, Software, Expressing, Quantitative Proteomics, Western Blot, Control, Comparison

Journal: Redox biology

Article Title: Ubiquitination as a key regulatory mechanism for O 3 -induced cutaneous redox inflammasome activation.

doi: 10.1016/j.redox.2022.102440

Figure Lengend Snippet: Fig. 2. 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-1β 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-1β 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 soft ware 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 compari son test. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Coverslips were incubated with primary antibody ASC (Cat. NBP1-78977 Novus Biological, Littleton, CO, USA) 1:100, NLRP1 (sc-166368 Santa Cruz Biotechnology Inc., Dallas, TX, USA) 1:50, 4HNE (ab46545, Abcam, Cambridge, UK) 1:400, DPP9 (ab42080, Abcam, Cambridge, UK)1:400, UBR2 (18852-1-AP, Proteintech Group Inc., Rosemont, IL, USA) 1: 150 in 0.25% BSA/PBS overnight at 4◦C.

Techniques: Activation Assay, Expressing, Double Immunofluorescence Staining, Staining, Software, Western Blot, Control

Journal: Redox biology

Article Title: Ubiquitination as a key regulatory mechanism for O 3 -induced cutaneous redox inflammasome activation.

doi: 10.1016/j.redox.2022.102440

Figure Lengend Snippet: Fig. 4. NLRP1 activation is mediated by its ubiq uitination. 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) repre sents 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 stain ing (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 soft ware and β-actin or Red ponceau were used as inter nal 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.)

Article Snippet: Coverslips were incubated with primary antibody ASC (Cat. NBP1-78977 Novus Biological, Littleton, CO, USA) 1:100, NLRP1 (sc-166368 Santa Cruz Biotechnology Inc., Dallas, TX, USA) 1:50, 4HNE (ab46545, Abcam, Cambridge, UK) 1:400, DPP9 (ab42080, Abcam, Cambridge, UK)1:400, UBR2 (18852-1-AP, Proteintech Group Inc., Rosemont, IL, USA) 1: 150 in 0.25% BSA/PBS overnight at 4◦C.

Techniques: Activation Assay, Expressing, Double Immunofluorescence Staining, Staining, Software, Western Blot, Control, Comparison