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: Membranes were incubated overnight at 4◦C with primary antibodies ASC (Cat. NBP1-78977 NovusBio, USA) 1:1000, Caspase 1 (2225S Cell Signaling Technology, Danvers, MA, USA) 1:1000, NLRP1 (AB-84361, SIC, Rome, Italy) 1:1000, Ubiquitin (ab7780, Abcam, USA) 1:2000, DPP9 (ab42080, Abcam, USA) 1:1000, 4HNE (ab46545, Abcam, USA), UBR2 (18852-1-AP Proteintech, USA) in TBS-T with 1% non-fat milk (Bio-Rad Laboratories, Inc., USA).

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: Membranes were incubated overnight at 4◦C with primary antibodies ASC (Cat. NBP1-78977 NovusBio, USA) 1:1000, Caspase 1 (2225S Cell Signaling Technology, Danvers, MA, USA) 1:1000, NLRP1 (AB-84361, SIC, Rome, Italy) 1:1000, Ubiquitin (ab7780, Abcam, USA) 1:2000, DPP9 (ab42080, Abcam, USA) 1:1000, 4HNE (ab46545, Abcam, USA), UBR2 (18852-1-AP Proteintech, USA) in TBS-T with 1% non-fat milk (Bio-Rad Laboratories, Inc., USA).

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. 3. Involvement of 4HNE protein adducts in NLRP1 inflammasome activation (a) 4HNE protein expression levels in HaCaT cells exposed at the indi cated doses of O3 for 1 h and collected right after the end of O3 exposure. (b) Immunoprecipitation assay for NLRP1 probed with 4HNE in HaCaT cells pre- treated with proteasome inhibitor MG-132 20 μM for 2 h and exposed to O3 or H2O2 50 μM for 1 h. Samples were collected right after the end of 1-h O3/ H2O2 exposures. (c) Double Immunofluorescence staining for NLRP1 (red) and 4HNE (green) in HaCaT cells after O3 exposure. The blue staining (DAPI) represents nuclei. Images were taken at 60 × magnification (scale bar = 10 μm) and the fluorescent levels were quantified using ImageJ software. (d) Quantification of protein expression levels of ubiq uitinated proteins in HaCaT cells exposed to O3. (e) Protein expression levels of ubiquitinated-NLRP1 after Immunoprecipitation for NLRP1 in HaCaT cells pre-treated with proteasome inhibitor MG-132 20 μM for 2 h and Cat 1000 U/ml for 2 h and exposed to O3 right after the end of exposure. For all the experi ments 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 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: Membranes were incubated overnight at 4◦C with primary antibodies ASC (Cat. NBP1-78977 NovusBio, USA) 1:1000, Caspase 1 (2225S Cell Signaling Technology, Danvers, MA, USA) 1:1000, NLRP1 (AB-84361, SIC, Rome, Italy) 1:1000, Ubiquitin (ab7780, Abcam, USA) 1:2000, DPP9 (ab42080, Abcam, USA) 1:1000, 4HNE (ab46545, Abcam, USA), UBR2 (18852-1-AP Proteintech, USA) in TBS-T with 1% non-fat milk (Bio-Rad Laboratories, Inc., USA).

Techniques: Activation Assay, Expressing, Immunoprecipitation, 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: Membranes were incubated overnight at 4◦C with primary antibodies ASC (Cat. NBP1-78977 NovusBio, USA) 1:1000, Caspase 1 (2225S Cell Signaling Technology, Danvers, MA, USA) 1:1000, NLRP1 (AB-84361, SIC, Rome, Italy) 1:1000, Ubiquitin (ab7780, Abcam, USA) 1:2000, DPP9 (ab42080, Abcam, USA) 1:1000, 4HNE (ab46545, Abcam, USA), UBR2 (18852-1-AP Proteintech, USA) in TBS-T with 1% non-fat milk (Bio-Rad Laboratories, Inc., USA).

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

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Siglec-15 is a putative receptor for porcine epidemic diarrhea virus infection

doi: 10.1007/s00018-025-05672-2

Figure Lengend Snippet: Expression and purification of H-Siglec-15 (20-263aa). ( A ) Schematic representation of the plasmids used for the expression of H-Siglec-15 (20-263aa)-Fc. ( B ) The expression of H-Siglec-15 (20-263aa)-Fc (approximately 220 kDa) in the culture supernatant of Expi293F cells was analyzed by SDS-PAGE and Coomassie blue staining. Samples 1–4 represent the culture supernatants collected on the third, fourth, fifth, and sixth days posttransfection, respectively. Sample 5 included the culture supernatant and cell precipitate from the sixth day, whereas samples 6 and 7 included the flow-through and elution of H-Siglec-15 (20-263aa)-Fc, respectively. Sample 8 was the cleaning solution, and sample 9 was the protein concentrate. M denotes the molecular weight marker. ( C ) Western blot analysis of H-Siglec-15 (20-263aa)-Fc expression in the culture supernatant of Expi293F cells. Samples 1–9 correspond to the same samples as in B , with M indicating the molecular weight marker. ( D ) The expression of TEV protease (approximately 30 kDa) was detected by SDS-PAGE and Coomassie blue staining. Samples 10 and 11 represent the precipitate and supernatant following cell lysis, whereas samples 12, 13, 14, and 15 represent the flow-through and elution at 20 mM, 80 mM, and 200 mM imidazole, respectively. M denotes the molecular weight marker. ( E ) Cleavage of the H-Siglec-15 (20-263aa)-Fc protein by the TEV protease was analyzed via SDS-PAGE and Coomassie blue staining. Sample 16 contains the uncleaved H-Siglec-15 (20-263aa)-Fc protein (220 kDa), whereas sample 17 contains the cleaved H-Siglec-15 (20-263aa) protein (approximately 27 kDa). Sample 18 was the concentrated cleaved H-Siglec-15 (20-263aa) protein. M denotes the molecular weight marker. ( F ) Cleavage of the H-Siglec-15 (20-263aa)-Fc protein by the TEV protease was confirmed by Western blot analysis. Samples 19–21 correspond to the same samples as in E , with M indicating the molecular weight marker

Article Snippet: The helper plasmids pCMV-VSV-G (#8454) and pSPAX2 for packaging lentivirus (#12260), the plasmid pX459 (#48139) for Siglec-15 knockout and the plasmid expressing TEV protease (pET28-MBP-super TEV protease) (#171782) were obtained from Addgene (Boston, MA, USA).

Techniques: Expressing, Purification, SDS Page, Staining, Molecular Weight, Marker, Western Blot, Lysis