Journal: Frontiers in Molecular Neuroscience

Article Title: Inflammasome Activation Induces Pyroptosis in the Retina Exposed to Ocular Hypertension Injury

doi: 10.3389/fnmol.2019.00036

Figure Lengend Snippet: Inflammasome activation after ocular hypertension (OHT) injury. (A) IL-1β cytokine release from OHT-challenged retina measured in naïve eyes, normotensive control (dotted lines) and experimental eyes (solid lines) at 6, 12 h and 24 h after injury in C57Bl6/J [Wild-type (WT), red], Panx1 −/− (Px1KO, blue) and Casp1 −/− Casp4(11) del (CaspDKO, dotted yellow) mice. Significant IL-1β release from control normotensive control eyes was only detected in WT animals (WT Cntr, dashed) at 6 h post-OHT; the zero level release from Px1KO and CaspDKO is shown as green dotted line. Gray dotted line represents limit of detection (LOD)/limit of quantification (LOQ) value of 0.58 ± 0.036 for these series of experiments. Statistics: mean ± standard deviation (SD); * P < 0.05; ns, no significance; N = 6. (B) Western blot analysis of NLRP3, Casp1 and gasdermin D proteins in control and experimental WT retinas at 6, 12 and 24 h post-OHT retinas. Untrimmed gel images: . (C) Changes in GSDMD gene expression in post-OHT retinas at 6, 12 and 24 h postinjury was assessed by qRT-PCR relative to naïve and normotensive control (cntr) retinas ( N = 3). Results are presented as fold change in Gsdmd transcript abundance, normalization to Gapdh. Statistics: mean ± SD, ** P < 0.01, T -Test; ns, non-significant changes.

Article Snippet: The following commercially available antibodies were used: neuronal Brn3a (Santa Cruz), class III β-tubulin (TUJ-1, Covance ID # MMS-435P), RBPMS (GeneTex ID# GTX118619), NeuN (Invitrogen ID#702022); glutamine synthetase (GeneTex ID# GTX109121); IL-1b (Cell Signaling ID#8689); Iba1 (Wako/FUJI ID# 019-19741), GFAP (Dako, cat#z0334), CD11b (Biolegend ID#101218), Casp1 (Novus Biologicals ID#NB100-56565R), Casp11 (Novus Biologicals ID# NB120-10454), ASC (Adipogen ID#AG25b-0006), NLRP1 (Novus Biologicals ID #NB100-56148), NLRP3 (Adipogen ID# AG20b-0014-C100), Aim2 (Boster Biological Technology ID#PB9683), GSDMD (Santa Cruz ID#sc-393656).

Techniques: Activation Assay, Control, Standard Deviation, Western Blot, Gene Expression, Quantitative RT-PCR

Journal: Frontiers in Molecular Neuroscience

Article Title: Inflammasome Activation Induces Pyroptosis in the Retina Exposed to Ocular Hypertension Injury

doi: 10.3389/fnmol.2019.00036

Figure Lengend Snippet: Activity of Casp1 in OHT-injured and normotensive control eyes. (A) Casp1 was detected by intraocular injection FLICA660-labeled substrate (green) in vivo 24 h after injury. Bright labeling (arrows) is evident in cells in the GCL and inner nuclear layer (INL) layers of the OHT-challenged retinas, a diffuse labeling of cell processes located in the IPL. Casp1 activity is diminished in Panx1 −/− (Px1 −/− OHT) retinas and WT retinas treated with probenecid (WT/Pbcd) at 12 h postinjury. (B) The analysis of the Casp1 immunolabeling (red, white arrows) in normotensive (NT control) and injured (OHT) retinas at 12 h and 24 h postinjury. Yellow arrows denote Casp1 colocalization with RGCs (RBPMS + , green) as well as with other cells at 24 h post-OHT. Bar, 25 μm.

Article Snippet: The following commercially available antibodies were used: neuronal Brn3a (Santa Cruz), class III β-tubulin (TUJ-1, Covance ID # MMS-435P), RBPMS (GeneTex ID# GTX118619), NeuN (Invitrogen ID#702022); glutamine synthetase (GeneTex ID# GTX109121); IL-1b (Cell Signaling ID#8689); Iba1 (Wako/FUJI ID# 019-19741), GFAP (Dako, cat#z0334), CD11b (Biolegend ID#101218), Casp1 (Novus Biologicals ID#NB100-56565R), Casp11 (Novus Biologicals ID# NB120-10454), ASC (Adipogen ID#AG25b-0006), NLRP1 (Novus Biologicals ID #NB100-56148), NLRP3 (Adipogen ID# AG20b-0014-C100), Aim2 (Boster Biological Technology ID#PB9683), GSDMD (Santa Cruz ID#sc-393656).

Techniques: Activity Assay, Control, Injection, Labeling, In Vivo, Immunolabeling

Journal: Frontiers in Molecular Neuroscience

Article Title: Inflammasome Activation Induces Pyroptosis in the Retina Exposed to Ocular Hypertension Injury

doi: 10.3389/fnmol.2019.00036

Figure Lengend Snippet: Post-OHT activation of gasdermin D (GSDMD) in the GCL. (A) Cytosolic labeling for gasdermin D co-localized with RGCs (RBPMS, white). At 12 h postinjury no co-localization was detected with GFAP + astrocytes (As, red, yellow arrows) or CD11b + microglia (MG, white, white arrows). (B) At 24 h post-OHT only low-grade GSDMD + Casp1 + cells with altered morphology, condensed or lacking nuclei are observed (yellow arrows), some retaining weak RBPMS marker labeling (white).GSDMD staining is mostly grainy. (C) GSDMD + cells (yellow arrows) are observed in close vicinity of astrocytes only in OHT injured but not in control retinas. (D) Inset shows a high resolution image of grainy GSDMD + labeling in neurons with altered morphology, condensed or lacking nuclei (yellow arrows) at 24 h post OHT. Astrocytes (red) in vicinity of GSDMD + neurons are highly positive for Casp1. Bar, 25 μm on all panels.

Article Snippet: The following commercially available antibodies were used: neuronal Brn3a (Santa Cruz), class III β-tubulin (TUJ-1, Covance ID # MMS-435P), RBPMS (GeneTex ID# GTX118619), NeuN (Invitrogen ID#702022); glutamine synthetase (GeneTex ID# GTX109121); IL-1b (Cell Signaling ID#8689); Iba1 (Wako/FUJI ID# 019-19741), GFAP (Dako, cat#z0334), CD11b (Biolegend ID#101218), Casp1 (Novus Biologicals ID#NB100-56565R), Casp11 (Novus Biologicals ID# NB120-10454), ASC (Adipogen ID#AG25b-0006), NLRP1 (Novus Biologicals ID #NB100-56148), NLRP3 (Adipogen ID# AG20b-0014-C100), Aim2 (Boster Biological Technology ID#PB9683), GSDMD (Santa Cruz ID#sc-393656).

Techniques: Activation Assay, Labeling, Marker, Staining, Control

Journal: Nature immunology

Article Title: SERPINB1-mediated checkpoint of inflammatory caspase activation

doi: 10.1038/s41590-018-0303-z

Figure Lengend Snippet: a, Identification of SERPINB1 as a binding partner of caspase-4 CARD (aa 2–124) by yeast two-hybrid screening. For yeast co-transformation assays, full-length and truncated forms of caspase-1/–4/–5 were co-transformed with the SERPINB1 carboxy-terminal region (aa 330–379) to yeast for growth on two dropout (DO) or four dropout containing X-α-Gal plates. b, Specific interaction between SERPINB1 and inflammatory caspase family members. GST–SERPINB1–330–379 and hemagglutinin (HA)-tagged CARD-containing caspases were transfected into 293 T cells, and whole-cell extracts (WCEs) were subjected to GST pulldown (GST-PD), followed by immunoblotting (IB) using anti-HA or anti-GST. c, Endogenous interaction between caspase-1/–4 and SERPINB1. THP1 WCEs were subjected to co-immunoprecipitation (IP) with control anti-HA or anti-caspase-1. U937 WCEs were subjected to co-immunoprecipitation with control anti-HA or anti-caspase-4. mAb and rAb denote mouse and rabbit antibodies, respectively. HC, heavy chain. d, Specific binding of caspase-4 to SERPINB1. HA-tagged caspase-4 and Flag-tagged serpin family members were transfected into 293 T cells, and WCEs were subjected to co-immunoprecipitation with anti-Flag, followed by immunoblotting using anti-HA or anti-Flag. e, Interaction of enzymatically inactive caspase mutants with SERPINB1. HA-tagged caspase-1 or caspase-4 wild-type or enzymatically inactive mutants and Flag-tagged SERPINB1 were transfected into 293 T cells, and WCEs were subjected to co-immunoprecipitation with anti-HA, followed by immunoblotting with anti-Flag or anti-HA. Data in a–e are representative of two independent experiments.

Article Snippet: Primary antibodies include antibodies to human SERPINB1 (3B4, Origene), human caspase-1 (14F468, Santa Cruz), human caspase-1 ( {"type":"entrez-protein","attrs":{"text":"EPR16883","term_id":"523382955","term_text":"EPR16883"}} EPR16883 , Abcam), human caspase-1 p20 (Bally-1, Adipogen), human caspase-1 CARD (A-19, Santa Cruz), mouse caspase-1 (M-20, Santa Cruz), human caspase-4 (4B9, Santa Cruz), caspase-2 (611022, BD), caspase-3 (8G10, Cell Signaling), IL-1β (2002, Cell Signaling), neutrophil elastase (C-17, Santa Cruz), proteinase-3 (684042, R&D Systems), cathepsin G (N-19, Santa Cruz), NLRP3 (Cryo-2, Adipogen), ASC (F-9, Santa Cruz), SMT3 (y-84, Santa Cruz), His (H-15, Santa Cruz), GST (B-14, Santa Cruz), Actin (C4, Santa Cruz), rabbit-Flag (F7425, Sigma), mouse-Flag (F1804, Sigma), rabbit-HA (PRB-101P, Covance), mouse-HA (16B12, BioLegend), mouse IgG-HRP (7076, Cell Signaling), rabbit IgG-HRP (7074, Cell Signaling) and goat IgG-HRP (sc-2020, Santa Cruz).

Techniques: Binding Assay, Two Hybrid Screening, Transformation Assay, Transfection, Western Blot, Immunoprecipitation

Journal: Nature immunology

Article Title: SERPINB1-mediated checkpoint of inflammatory caspase activation

doi: 10.1038/s41590-018-0303-z

Figure Lengend Snippet: a, Schematic diagram of SERPINB1 truncation and alanine-substitution mutant constructs. The relative binding intensity is indicated as + ++ > ++ > + > (+ ) > −. b,c, GST-pulldown assay of SERPINB1 truncation mutants binding to caspase-1 and caspase-4. GST-SERPINB1 truncated forms and caspase-1-C285A–HA or caspase-4-C258A–HA were transfected into 293 T cells, and WCEs were subjected to GST pulldown, followed by immunoblotting using anti-HA or anti-GST. d, GST-pulldown assay of SERPINB1 alanine-substitution mutants binding to caspase-4. GST-SERPINB1 truncated or alanine-substituted forms and caspase-4-C258A–HA were transfected into 293 T cells, and WCEs were subjected to GST pulldown, followed by immunoblotting using anti-HA or anti-GST. Data in b–d are representative of two independent experiments.

Article Snippet: Primary antibodies include antibodies to human SERPINB1 (3B4, Origene), human caspase-1 (14F468, Santa Cruz), human caspase-1 ( {"type":"entrez-protein","attrs":{"text":"EPR16883","term_id":"523382955","term_text":"EPR16883"}} EPR16883 , Abcam), human caspase-1 p20 (Bally-1, Adipogen), human caspase-1 CARD (A-19, Santa Cruz), mouse caspase-1 (M-20, Santa Cruz), human caspase-4 (4B9, Santa Cruz), caspase-2 (611022, BD), caspase-3 (8G10, Cell Signaling), IL-1β (2002, Cell Signaling), neutrophil elastase (C-17, Santa Cruz), proteinase-3 (684042, R&D Systems), cathepsin G (N-19, Santa Cruz), NLRP3 (Cryo-2, Adipogen), ASC (F-9, Santa Cruz), SMT3 (y-84, Santa Cruz), His (H-15, Santa Cruz), GST (B-14, Santa Cruz), Actin (C4, Santa Cruz), rabbit-Flag (F7425, Sigma), mouse-Flag (F1804, Sigma), rabbit-HA (PRB-101P, Covance), mouse-HA (16B12, BioLegend), mouse IgG-HRP (7076, Cell Signaling), rabbit IgG-HRP (7074, Cell Signaling) and goat IgG-HRP (sc-2020, Santa Cruz).

Techniques: Mutagenesis, Construct, Binding Assay, GST Pulldown Assay, Transfection, Western Blot

Journal: Nature immunology

Article Title: SERPINB1-mediated checkpoint of inflammatory caspase activation

doi: 10.1038/s41590-018-0303-z

Figure Lengend Snippet: a, IL-1β secretion after SERPINB1 depletion. THP1 cells were transduced with scrambled or SERPINB1-specific shRNA (shSERPINB1) lentivirus for 48 h and were primed with LPS (1 μg ml−1) for 12 h. b, Immunoblots of the secreted p17 form of IL-1β after SERPINB1 depletion. c, Effects of deletion of the gene encoding caspase-1 or caspase-4 on SERPINB1-depletion-induced IL-1β secretion. d, Immunoblots of the cleaved p20 form of caspase-1 and processed p17 form of IL-1β in THP1-sgNT or THP1-sgCASP1 cells after SERPINB1 depletion. e,f, Effects of knockdown of genes encoding neutrophil elastase (shNE), proteinase-3 (shPRTN3), cathepsin G (shCG), ASC (shASC) or NLRP3 (shNLRP3) on SERPINB1-depletion-induced IL-1β secretion. g, Effects of caspase inhibitors on SERPINB1-depletion-induced cell death. SYTOX Green–based cell death was measured without or with LPS priming. h, Transmission electron microscopic images of U937 cells after SERPINB1 depletion. For a positive control, cells were electroporated with 1 μg LPS. Large vacuoles (filled triangles), disrupted nucleus (asterisks), swollen mitochondria (arrows). Scale bars, 1 μm. N, nucleus; V, phagocytic vacuole; M, mitochondria. Data in b,d,g,h are representative of two independent experiments. Whole-cell lysates (WCLs) and supernatants (SUPs) in b,d were immunoblotted with indicated antibodies. Data are presented as mean ± s.e.m. from n = 3 independent experiments in a,e,f and from n = 5 independent experiments in c, and as box and whiskers (min to max) from n = 3 technical replicates in g. P values were determined by one-way analysis of variance (ANOVA) with Dunnett’s comparison relative to scramble in a and by two-way ANOVA with Bonferroni’s comparison relative to non-targeting control (sgNT) in c, to scramble in e,f, or to dimethylsulfoxide (DMSO) in g. NS, not significant.

Article Snippet: Primary antibodies include antibodies to human SERPINB1 (3B4, Origene), human caspase-1 (14F468, Santa Cruz), human caspase-1 ( {"type":"entrez-protein","attrs":{"text":"EPR16883","term_id":"523382955","term_text":"EPR16883"}} EPR16883 , Abcam), human caspase-1 p20 (Bally-1, Adipogen), human caspase-1 CARD (A-19, Santa Cruz), mouse caspase-1 (M-20, Santa Cruz), human caspase-4 (4B9, Santa Cruz), caspase-2 (611022, BD), caspase-3 (8G10, Cell Signaling), IL-1β (2002, Cell Signaling), neutrophil elastase (C-17, Santa Cruz), proteinase-3 (684042, R&D Systems), cathepsin G (N-19, Santa Cruz), NLRP3 (Cryo-2, Adipogen), ASC (F-9, Santa Cruz), SMT3 (y-84, Santa Cruz), His (H-15, Santa Cruz), GST (B-14, Santa Cruz), Actin (C4, Santa Cruz), rabbit-Flag (F7425, Sigma), mouse-Flag (F1804, Sigma), rabbit-HA (PRB-101P, Covance), mouse-HA (16B12, BioLegend), mouse IgG-HRP (7076, Cell Signaling), rabbit IgG-HRP (7074, Cell Signaling) and goat IgG-HRP (sc-2020, Santa Cruz).

Techniques: Transduction, shRNA, Western Blot, Transmission Assay, Positive Control

Journal: Nature immunology

Article Title: SERPINB1-mediated checkpoint of inflammatory caspase activation

doi: 10.1038/s41590-018-0303-z

Figure Lengend Snippet: a, The RCL and CBM sequence alignment of human SERPINB1 and mouse SERPINB1a, SERPINB1b and SERPINB1c. The conserved P2–P1–P1 residues in the RCL sequence are indicated by orange font, and the CBM sequence is shown in red font. The conserved 13 aa in the CBM sequence are indicated in the red-dotted box. Specified amino acid numbers are based on human SERPINB1. b, Verification of Serpinb1-targeting shRNA’s silencing efficiency by qRT-PCR in DC2.4 cells. mRNA expression was normalized to 18S, and fold change was calculated relative to scramble. c, Immunoblots of cleaved p10 subunit of caspase-1 in DC2.4 cells after Serpinb1a, Serpinb1b and/or Serpinb1c depletion. WCLs and SUPs were obtained at 48 h post-transduction and were immunoblotted with indicated antibodies. d, Caspase-1 activation in Serpinb1a−/− pPMNs. FLICA-positive staining was analyzed by flow cytometry. e,f, IL-1β secretion and cell death after Serpinb1 depletion in BMDMs. Wild-type (WT) and Casp1−/−Casp11−/− BMDMs were transduced with scrambled or pan-Serpinb1 shRNA lentivirus. Cytokines were quantified by enzyme-linked immunosorbent assay (ELISA), and cell viability was determined by ATP-based assay. Data in c are representative of two independent experiments. Data are presented as mean ± s.e.m. from n = 3 independent experiments in b,f and from n = 7 per group, pooled from two independent experiments in d, and as box and whiskers (min to max) from n = 6 pooled from three independent experiments in e. P values were determined by one-way ANOVA with Dunnett’s comparison relative to scramble in b, by an two-tailed unpaired t-test in d and by two-way ANOVA with Bonferroni’s comparison relative to scramble in e,f.

Article Snippet: Primary antibodies include antibodies to human SERPINB1 (3B4, Origene), human caspase-1 (14F468, Santa Cruz), human caspase-1 ( {"type":"entrez-protein","attrs":{"text":"EPR16883","term_id":"523382955","term_text":"EPR16883"}} EPR16883 , Abcam), human caspase-1 p20 (Bally-1, Adipogen), human caspase-1 CARD (A-19, Santa Cruz), mouse caspase-1 (M-20, Santa Cruz), human caspase-4 (4B9, Santa Cruz), caspase-2 (611022, BD), caspase-3 (8G10, Cell Signaling), IL-1β (2002, Cell Signaling), neutrophil elastase (C-17, Santa Cruz), proteinase-3 (684042, R&D Systems), cathepsin G (N-19, Santa Cruz), NLRP3 (Cryo-2, Adipogen), ASC (F-9, Santa Cruz), SMT3 (y-84, Santa Cruz), His (H-15, Santa Cruz), GST (B-14, Santa Cruz), Actin (C4, Santa Cruz), rabbit-Flag (F7425, Sigma), mouse-Flag (F1804, Sigma), rabbit-HA (PRB-101P, Covance), mouse-HA (16B12, BioLegend), mouse IgG-HRP (7076, Cell Signaling), rabbit IgG-HRP (7074, Cell Signaling) and goat IgG-HRP (sc-2020, Santa Cruz).

Techniques: Sequencing, Quantitative RT-PCR, Expressing, Western Blot, Transduction, Activation Assay, Staining, Flow Cytometry, shRNA, Enzyme-linked Immunosorbent Assay, ATP Assay, Two Tailed Test

Journal: Nature immunology

Article Title: SERPINB1-mediated checkpoint of inflammatory caspase activation

doi: 10.1038/s41590-018-0303-z

Figure Lengend Snippet: a, Increased oligomerization of endogenous caspase-1/–4 after SERPINB1 depletion. Scramble or shSERPINB1 lentivirus–infected THP1 cells were pre-incubated with z-VAD-FMK and LPS for 6 h before DSS crosslinking. Crosslinked cells were lysed, and WCEs were immunoblotted with indicated antibodies. Square bracket (right margin) indicates oligomerized caspase-1 or caspase-4. b, Size-exclusion chromatography assay of caspase-1 CARD oligomerization. MBP-1CARD-SUMO was incubated with or without SUMO-SERPINB1 at 4 °C for 12 h and subjected to TEV cleavage. The indicated samples were analyzed by size-exclusion columns. SDS-PAGE of each fraction was stained with Coomassie blue. The red-dotted box indicates the fractions containing 1CARD-SUMO proteins. c, SERPINB1-mediated inhibition of caspase-1 CARD oligomerization. MBP-1CARD-SUMO (100 nM) was incubated with or without SERPINB1 (100 nM) at 4 °C for 12 h and was subjected to TEV cleavage for 2 h, followed by BS3 crosslinking. Crosslinked proteins were immunoblotted with indicated antibodies. Relative density was calculated by Image Lab Software. d,e, Fluorescence polarization assay of caspase-1 CARD oligomerization. SERPINB1 was incubated with FITC-conjugated MBP-1CARD-SUMO or together with MBP-ASC at 4 °C for 12 h and was subjected to TEV cleavage. The fluorescence polarization signals were collected at the indicated time point with an Envision plate reader. Data in a–e are representative of two independent experiments. Data in d,e are presented as mean ± s.e.m. from n = 3 technical replicates.

Article Snippet: Primary antibodies include antibodies to human SERPINB1 (3B4, Origene), human caspase-1 (14F468, Santa Cruz), human caspase-1 ( {"type":"entrez-protein","attrs":{"text":"EPR16883","term_id":"523382955","term_text":"EPR16883"}} EPR16883 , Abcam), human caspase-1 p20 (Bally-1, Adipogen), human caspase-1 CARD (A-19, Santa Cruz), mouse caspase-1 (M-20, Santa Cruz), human caspase-4 (4B9, Santa Cruz), caspase-2 (611022, BD), caspase-3 (8G10, Cell Signaling), IL-1β (2002, Cell Signaling), neutrophil elastase (C-17, Santa Cruz), proteinase-3 (684042, R&D Systems), cathepsin G (N-19, Santa Cruz), NLRP3 (Cryo-2, Adipogen), ASC (F-9, Santa Cruz), SMT3 (y-84, Santa Cruz), His (H-15, Santa Cruz), GST (B-14, Santa Cruz), Actin (C4, Santa Cruz), rabbit-Flag (F7425, Sigma), mouse-Flag (F1804, Sigma), rabbit-HA (PRB-101P, Covance), mouse-HA (16B12, BioLegend), mouse IgG-HRP (7076, Cell Signaling), rabbit IgG-HRP (7074, Cell Signaling) and goat IgG-HRP (sc-2020, Santa Cruz).

Techniques: Infection, Incubation, Size-exclusion Chromatography, SDS Page, Staining, Inhibition, Software, Fluorescence