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

Journal: bioRxiv

Article Title: Recessive POPDC1 Truncation Causes Lethal Short-QT Pattern Arrhythmogenic Cardiomyopathy with Multi-Ion Channel Remodeling and Ankyrin-G Scaffold Disruption

doi: 10.64898/2026.03.07.710328

Figure Lengend Snippet: (A) Volcano plot depiction of the differential proteome from ventricular tissue lysates of Popdc1 +/+ and Popdc1 fs/fs rats. Prioritization of the top targets highlighted profound downregulation of BVES (POPDC1) and the membrane scaffold Ankyrin-G (Ank3) (n=5-7). (B) KEGG pathway enrichment of the proteome identified “Cardiac muscle contraction” and “Arrhythmogenic right ventricular cardiomyopathy” and various cardiomyopathies as the top disrupted pathways. (C) Immunofluorescence staining of the myocardium demonstrates the loss of both BVES and Ankyrin-G (both in red) in Popdc1 fs/fs rats. (D) RT-qPCR gene expression analyses in cardiac tissue demonstrated reduced Popdc1 but normalized AnkG mRNA (n=5-7). (E) Western blots confirm the reduction of cardiac POPDC1-Ankyrin-G-Nav1.5 axis and TREK-1 (KCNK2), concurrent with an upregulation of Kv4.3 and Cav1.2 in Popdc1 fs/fs rats (n=6). (F–G) Popdc1 fs/fs hearts exhibit markers of oxidative stress: elevated malondialdehyde (MDA; G) and reduced superoxide dismutase (SOD; H) activity (n=4). (F) Transmission electron microscopy (TEM) displays widened intercalated discs and myofibrillar disarray (red arrowhead) in Popdc1 fs/fs hearts (n=3). Data are mean ± SD. Statistical analyses conducted were unpaired Student’s t-test. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 respectively.

Article Snippet: Blots were probed with primary antibodies against Cav1.2 (1:1000, MA5-45389, Thermo Fisher Scientific), Kv4.3 (1:500, PA5-93292, Thermo Fisher Scientific), Nav1.5 (1:200, ASC-005, Alomone Labs), TREK-1/KCNK2 (1:1000, PA5-115452, Thermo Fisher Scientific), AnkG (1:1000, 27980-1-AP, Proteintech), BVES (1:1000, as above, Thermo Fisher Scientific), and GAPDH (1:10000, 60004-1-Ig, Proteintech).

Techniques: Membrane, Immunofluorescence, Staining, Quantitative RT-PCR, Gene Expression, Western Blot, Activity Assay, Transmission Assay, Electron Microscopy

Journal: Journal of cellular and molecular medicine

Article Title: Tojapride prevents CaSR-mediated NLRP3 inflammasome activation in oesophageal epithelium irritated by acidic bile salts.

doi: 10.1111/jcmm.14631

Figure Lengend Snippet: FIGURE 4 CaSR expression and colocalization of NLRP3-ASC in the oesophagus of modified RE rats. A, Enzyme-linked immunohistochemistry in paraffin-embedded sections of oesophageal tissue using a mouse monoclonal CaSR antibody. Scale bar: 200 μm. B, Immunofluorescence in paraffin-embedded sections of oesophageal tissue using goat polyclonal NLRP3 (green) and rabbit polyclonal ASC (red) antibodies. Scale bar: 100 μm. The yellow area in the basal cells of the epithelium indicates the colocalization of the green and red channels

Article Snippet: After blocking with 5% skim milk for 1 hour at room temperature, the membranes were incubated with the following primary antibodies: CaSR (1:200, ab19347, Abcam), NLRP3 (1:100, ab214185, Abcam), ASC (1:100, sc-22514-R, Santa Cruz), Caspase-1 p20 (1:100, sc-398715, Santa Cruz) and IL-1β (1:100, sc-32294, Santa Cruz) at 4°C overnight.

Techniques: Expressing, Modification, Immunohistochemistry, Immunofluorescence