Journal: Cell Death Discovery

Article Title: Ablation of Cbl-b and c-Cbl in dendritic cells causes spontaneous liver cirrhosis via altering multiple properties of CD103 + cDC1s

doi: 10.1038/s41420-022-00953-2

Figure Lengend Snippet: A CD103 + cDCs (MHC-II + CD11c + ) differentiated using 20 ng/mL Flt3-L and 2 ng/mL GM-CSF were sorted and protein levels of caspase-8 and caspase-9 were measured by Western blot. B Quantitative RT-PCR analysis of Bim mRNA levels in BM-derived CD103 + cDCs. n = 3 per group. C Western blot analysis of Bim expression in BM-derived CD103 + cDCs. D Quantitative RT-PCR analysis of Bcl-2 and Bcl-xL mRNA levels in BM-derived CD103 + cDCs. n = 3 per group. E Western blot analysis of Bcl-2 and Bcl-xL expression levels in BM-derived CD103 + cDCs. F Western blot analysis of STAT5 and STAT3 expression levels in BM-derived CD103 + cDCs. G STAT5-IN-1 was added to CD103 + cDC cultures at the indicated concentration for 9 days after which apoptosis levels were analyzed by flow cytometry. n = 4 per group. H The percentage of Annexin V + 7AAD − CD103 + cDCs treated with the indicated concentration of the STAT5 inhibitor. I Schematic of the Bim gene. Vertical boxes indicate exons and the location of conserved GAS sequences is shown. J Luciferase assays of STAT5 binding to the putative GAS sites. n = 4 per group. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns, no significance based on One-Way ANOVA comparisons. p < 0.05 was considered statistically significant.

Article Snippet: The following antibodies were used in our study: anti-Cbl-b (D3C12) (#9498), anti-c-Cbl (D4E10) (#8447), anti-caspase-3 (D3R6Y) (#14220), anti-caspase-7 (D2Q3L) (#12827), anti-caspase-8 (1C12) (#9746), anti-caspase-9 (C9) (#9508), anti-cleaved caspase-3 (Asp175) (#9661), anti-cleaved caspase-7 (D198) (#9491), anti-cleaved caspase-8 (clone D5B2) (#8592), anti-cleaved caspase-9 (D8I9E) (#20750), anti-Bim (C34C5) (#2933), anti-STAT3 (D3Z2G) (#12640), anti-STAT5 (D206Y) (#94205), anti-Bcl-2 (D17C4) (#3498), anti-Bcl-xL (54H6) (##2764), anti-Myc (9B11) (#2276) and anti-HA (C29F4) (#5017), all purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Western Blot, Quantitative RT-PCR, Derivative Assay, Expressing, Concentration Assay, Flow Cytometry, Luciferase, Binding Assay

Journal: EMBO Reports

Article Title: IKKβ is required for the formation of the NLRP3 inflammasome

doi: 10.15252/embr.202050743

Figure Lengend Snippet: WT BMDM were incubated for 1 h without (−) or with (+) 5 μM BI605906, 5 μM TPCA‐1 or 1 μM MCC950. The cells were then stimulated for 1 h with (+) 100 ng/ml LPS and/or 5 μM nigericin, or left unstimulated (−). After cell lysis in the presence of 1% (v/v) Triton X‐100, the Triton X‐100‐insoluble fractions were prepared as in Fig , denatured in SDS, subjected to SDS–PAGE and immunoblotted with anti‐ASC or with an antibody against the U2 small nuclear RNA auxiliary factor 1 (U2AF1) as a loading control. As in A, except that the Triton X‐100‐insoluble fraction was first crosslinked by incubation for 45 min at 37°C with 2.0 mM DSS. (A, B) Similar results were obtained in two independent experiments. WT BMDM were incubated for 1 h with (+) or without (−) the IKKβ inhibitors BI605906 (5 μM), TPCA‐1 (5 μM) or PS1145 (10 μM), the TAK1 inhibitor NG25 (2 μM) or the NLRP3 inflammasome inhibitor MCC950 (1 μM). The cells were then stimulated with LPS (100 ng/ml) and/or 5 μM nigericin (+) or left unstimulated (‐). Cells were stimulated with TNF (10 ng/ml) and cycloheximide (Chx) (10 μg/ml), which was used as a positive control for a signal generating cleaved (CL) caspase‐8. Cells were lysed, cell extracts (10 µg protein) were subjected to SDS–PAGE and immunoblotted with antibodies recognizing caspase‐8(CL) (the cleavage product of caspase‐8) and GAPDH. As in C, except that the samples were immunoblotted for full length caspase‐1 (caspase‐1), the p20 fragment of caspase‐1 (p20) and GAPDH. Similar results were obtained in two independent experiments. WT BMDM were stimulated for 4 h without (Panel 1, control) or with (Panels 2–5) 100 ng/ml LPS and then incubated for 1 h without (Panels 1–3) or with 5 µM TPCA‐1(Panel 4) or 5 µM BI605906 (Panel 5) and then stimulated for 60 min with 5 µM nigericin (Panels 3–5) or without nigericin (Panels 1 and 2). The cells were fixed and processed for immunofluorescence using a rabbit polyclonal antibody against TGN38, which was visualized using a secondary antibody (red). Nuclei were counterstained with DAPI (blue). Images were acquired by sequential laser scanning on the confocal microscope. Similar results were obtained in three independent experiments, and representative images are shown. Data information: In all panels, scale bar = 50 µm. Source data are available online for this figure.

Article Snippet: Rabbit monoclonal antibodies recognizing p105/NF‐κB1 phosphorylated at Ser933 (18E6), ERK1 and ERK2 phosphorylated at the Thr‐Glu‐Tyr motif in the activation loop (D13.14.4E), IRF3 phosphorylated at Ser396 (4D4G), the cleaved form of caspase‐8 (D5B2) and all forms of glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) (14C10) were from Cell Signalling Technology (CST).

Techniques: Incubation, Lysis, SDS Page, Positive Control, Immunofluorescence, Microscopy

Journal: Nature Communications

Article Title: TNF controls a speed-accuracy tradeoff in the cell death decision to restrict viral spread

doi: 10.1038/s41467-021-23195-9

Figure Lengend Snippet: A Quantification of cell death in NIH 3T3 fibroblasts after treatment with 100 ng/ml TNF or TNF supplemented with 0.25 μg/ml Actinomycin D. Cell death was quantified over time based on Hoechst 33342 fluorescence intensity which increases sharply on nuclear condensation. B Quantification of cell death in fibroblasts 24 h after simultaneous treatment with 0.3 ng/ml TNF and 50 pg/ml LCL-161. Cell death was quantified based on incorporation of a nucleic acid stain (sytox green). C Diagram of experiment for data shown in D . 3T3 fibroblasts were treated for 24 h with 0.3 ng/ml TNF and then washed and rested overnight. The next day, LCL-161 was added at the indicated doses and cell death was quantified after 24 h of LCL-161 exposure. D Quantification of cell death for experiment described in C . Cell death was quantified based on incorporation of sytox green. E Quantification of 3T3 fibroblasts that stained positive for cleaved Caspase-8 during and after treatment with 100 ng/ml TNF. The shaded region indicates the time period before TNF was washed out. Cleaved Caspase-8 was detected by immunostaining and cells were distinguished as positive by setting a threshold on the antibody fluorescence intensity. F Quantification of cell death for 3T3 fibroblasts treated constitutively with 30 ng/ml TNF or washed after overnight treatment with 30 ng/ml TNF. Cell death was quantified based on incorporation of sytox green. G Diagram of experiment for data shown in H . 3T3 fibroblasts were treated with 1 ng/ml TNF for 24 h and then washed. At different times after washing, 0.25 μg/ml Actinomycin D was added to cells for 4 h before quantifying cell death. H Quantification of cell death for experiment described in G . Cell death was quantified based on incorporation of sytox green. B , D , F bars denote the mean of replicates (filled black circles). E , H are mean ± s.d.

Article Snippet: The primary antibodies used were: Monoclonal anti-Cleaved Caspase-8 (clone D5B2) (Cell Signaling Technologies), monoclonal anti-Cleaved Caspase 3 (clone D175) (Cell Signaling Technologies), monoclonal anti-phospho-STING (clone D1C4T) (Cell Signaling Technologies), monoclonal anti-TNFα (clone MP6-XT22) (Biolegend), and monoclonal anti-Ki-67 (clone 16A8) (Biolegend).

Techniques: Fluorescence, Staining, Immunostaining

Journal: iScience

Article Title: NLRC4 inhibits NLRP3 inflammasome and abrogates effective antifungal CD8 + T cell responses

doi: 10.1016/j.isci.2021.102548

Figure Lengend Snippet: NLRC4 regulates early IL-1β release after P. brasiliensis infection WT and Nlrc4 −/− and mice were intravenously infected with 1x10 6 of viable Pb18 yeasts. (A) Scatterplots with bars show the fungal load in lung of WT and Nlrc4 −/− mice at 7 dpi. Results represent a pool of three independent experiments. (B) Scatterplots with bars show the quantification of IL-1β at 7 in the lungs of WT and Nlrc4 −/− mice. (C) Western blotting analysis detecting IL-1β cleavage (p17) in lung homogenates from WT and Nlrc4 −/− mice at 7 dpi. The intensities of IL-1β protein were quantified using iBright™ CL1500 Imaging System. (D) IL-1β expression was visualized in the lungs of mice at 7 dpi from both groups using immunohistochemistry (Scale bar: 50 μM – left panel). Each column represents the mean ± SD. (E) Caspase-1 expression was visualized in the lungs of mice at 7 dpi from both groups using immunohistochemistry (Scale bar: 20 μM – right panel). Each column represents the mean ± SD. (F) Active caspase-1 (p20) was detected by Western blotting in the lungs from WT and Nlc4 −/− mice at 7 dpi. The intensities of active caspase-1 were quantified using iBright™ CL1500 Imaging System. (G) Active caspase-8 (p18) was detect by Western blotting in the lungs from WT and Nlc4 −/− mice at 7 dpi. The intensities of active caspase-8 were quantified using iBright™ CL1500 Imaging System. (H) Stacked bars show the percentage of neutrophils (Ly6G + CD11b + ), monocytes (Ly6G − , Ly6C + CD11b + ), macrophages (CD11b + F4/80 + MHC-II + ), and dendritic cells (CD11c + CD11b + MHC-II + ). (I) Contour plots show representative flow cytometric data and scatterplots with bars show the absolute numbers of macrophages and dendritic cells gated in CD45 + IL-1β + cells at 7 dpi (n = 5 infected mice per group at 7 dpi).Data represent one of two independent experiments ( n = 3– 5 infected mice per group at each time point). Data are expressed as mean ± SD. Statistically significant differences were evaluated with ANOVA followed by Bonferroni's multiple comparisons test or unpaired t-test (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001). See also Figure S1 .

Article Snippet: Rabbit mAb cleaved Caspase-8 (Asp387) (D5B2) XP® , Cell Signaling Technology , Cat#8592S; RRID: AB_10891784.

Techniques: Infection, Western Blot, Imaging, Expressing, Immunohistochemistry

Journal: iScience

Article Title: NLRC4 inhibits NLRP3 inflammasome and abrogates effective antifungal CD8 + T cell responses

doi: 10.1016/j.isci.2021.102548

Figure Lengend Snippet:

Article Snippet: Rabbit mAb cleaved Caspase-8 (Asp387) (D5B2) XP® , Cell Signaling Technology , Cat#8592S; RRID: AB_10891784.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Bradford Protein Assay, Knock-Out, Double Knockout, Mouse Assay, Software, Imaging

Journal: Cellular and Molecular Neurobiology

Article Title: TNF-α Triggers RIP1/FADD/Caspase-8-Mediated Apoptosis of Astrocytes and RIP3/MLKL-Mediated Necroptosis of Neurons Induced by Angiostrongylus cantonensis Infection

doi: 10.1007/s10571-021-01063-w

Figure Lengend Snippet: Antibodies

Article Snippet: Cleaved caspase-8 (Asp387) (D5B2) XP® rabbit mAb (mouse specific) , Cell signaling technology , #8592.

Techniques:

Journal: Cellular and Molecular Neurobiology

Article Title: TNF-α Triggers RIP1/FADD/Caspase-8-Mediated Apoptosis of Astrocytes and RIP3/MLKL-Mediated Necroptosis of Neurons Induced by Angiostrongylus cantonensis Infection

doi: 10.1007/s10571-021-01063-w

Figure Lengend Snippet: The PPI network for the core enriched genes related to the apoptosis and necroptosis pathways. a , b . PPI network analysis for the core enriched genes of GSEA (as shown in Fig. c, d) showed that caspase-8 and TNF-α were the hub genes for the apoptosis and necroptosis pathways in mouse brains infected by AC, respectively. AC Angiostrongylus cantonensis

Article Snippet: Cleaved caspase-8 (Asp387) (D5B2) XP® rabbit mAb (mouse specific) , Cell signaling technology , #8592.

Techniques: Infection

Journal: Cellular and Molecular Neurobiology

Article Title: TNF-α Triggers RIP1/FADD/Caspase-8-Mediated Apoptosis of Astrocytes and RIP3/MLKL-Mediated Necroptosis of Neurons Induced by Angiostrongylus cantonensis Infection

doi: 10.1007/s10571-021-01063-w

Figure Lengend Snippet: TNF-α triggers RIP1/FADD/caspase-8-mediated apoptosis of astrocytes in mouse brains with AC infection. a Relative mRNA level of TNF-α in mouse brains upon AC infection was significantly higher than that in the control group. b TNF-α protein expression level in the mouse brain post infection was remarkably elevated, as revealed by immunofluorescence (left) and relative mean fluorescence intensity (MFI, right). c The lysates of brain tissues from normal control mice or mice post infection were subjected to western blot to determine the protein levels of genes related to the apoptosis signalling pathway (TNF-α, RIP1, caspase-8 and cleaved caspase-8). d – g The protein expression levels in c were quantified via density analysis. h Mice were infected by AC for 21 days and then the lysates of mouse brain tissues were immunoprecipitated with an anti-RIP1 antibody or an IgG antibody and the precipitated complexes were separately analysed by immunoblotting with antibodies against RIP1, RIP3, FADD, caspase-8 and GAPDH. i Cleaved caspase-3 and GFAP (specific marker of astrocytes) were co-localized, as shown by immunofluorescence. j Cleaved caspase-3 and NeuN (specific marker of neurons) showed no co-localization, as shown by immunofluorescence. * p < 0.05, ** p < 0.01, *** p < 0.001 (student’s t test). AC Angiostrongylus cantonensis , Ctrl normal control, INF infected by Angiostrongylus cantonensis

Article Snippet: Cleaved caspase-8 (Asp387) (D5B2) XP® rabbit mAb (mouse specific) , Cell signaling technology , #8592.

Techniques: Infection, Expressing, Immunofluorescence, Fluorescence, Western Blot, Immunoprecipitation, Marker

Journal: Immunity

Article Title: Flexible Usage and Interconnectivity of Diverse Cell Death Pathways Protect against Intracellular Infection

doi: 10.1016/j.immuni.2020.07.004

Figure Lengend Snippet: Combined Loss of Caspases-1, -11, -12, and -8 (plus RIPK3) Leads to Lack of Bacterial Control upon Salmonella Infection (A) Bacterial replication over time in WT and Casp1 –/– ;Casp11 –/– mice infected with Salmonella Δ AroA (200 CFU). n = 10−22 mice per group per time point. Mean and SEM are shown. ∗∗ p < 0.005, ∗ p < 0.05, ns p > 0.05 = not significant. (B) Bacterial loads in spleen and liver of mice of the indicated genotypes 3 weeks post-infection with Salmonella Δ AroA (200 CFU). n = 7−48 mice per genotype. Mean and SEM are shown. ∗∗ p < 0.005, ∗ p < 0.05, ns p > 0.05 = not significant. (C) Bacterial loads in spleen and liver from mice of the indicated genotypes 1 to 3 weeks post-infection with Salmonella Δ AroA (200 CFU). n = 3−4 mice per genotype and time point. Mean and SEM are shown. ∗∗ p < 0.005, ∗ p < 0.05, ns p > 0.05 = not significant. (D) Mouse survival curves and corresponding bacterial loads in the spleen and liver at time of sacrifice in WT and Casp1 –/– ;Casp11 –/– ;Casp12 –/– ;Casp8 –/– ;Ripk3 −/− mice infected with Salmonella Δ AroA (200 CFU). n = 7−8 mice per genotype. Mean and SEM are shown. ∗∗ p < 0.005. (E) Bone marrow chimeras of the indicated genotypes were infected with Salmonella Δ AroA (200 CFU) and culled for analysis of bacterial loads in spleen and liver 3 weeks post-infection. n = 10 mice per group. Mean and SEM are shown. ∗∗ p < 0.005. Please also see Figure S1 .

Article Snippet: rabbit anti-cleaved caspase-8 (D5B2) , CST , Cat# 8592S; RRID: AB_10891784.

Techniques: Infection

Journal: Immunity

Article Title: Flexible Usage and Interconnectivity of Diverse Cell Death Pathways Protect against Intracellular Infection

doi: 10.1016/j.immuni.2020.07.004

Figure Lengend Snippet: Caspase-8-Mediated Apoptosis Is the Default Backup Mechanism when Caspases-1- and 11-Mediated Pyroptosis Is Disabled in Salmonella -Infected iBMDMs (A) LDH release cell death assay of iBMDMs of the indicated genotypes after infection with Salmonella SL1344 (MOI = 50). Data pooled from two or more experiments. Mean and SEM are shown. ∗∗ p < 0.005, ns p > 0.05 = not significant. (B) Immunoblot analysis of the indicated proteins in iBMDMs of the indicated genotypes after infection with Salmonella SL1344 (MOI = 50). Probing for β-actin served as a loading control. (C) LDH release cell death assay of Salmonella SL1344 (MOI = 50) -infected WT and Casp1 –/– ;Casp11 –/– ;Casp12 –/– iBMDMs that had been left untreated or treated with the RIPK1 inhibitor, Nec1s (30 μM). Data pooled from two or more experiments. Mean and SEM are shown. ∗∗ p < 0.005, ns p > 0.05 = not significant. (D) Immunoblot analysis of the indicated proteins in Salmonella SL1344 (MOI = 50) -infected WT and Casp1 –/– ;Casp11 –/– ;Casp12 –/– iBMDMs that had been left untreated or treated with the RIPK1 inhibitor, Nec1s (30 μM). Probing for β-actin served as a loading control. Please see also and .

Article Snippet: rabbit anti-cleaved caspase-8 (D5B2) , CST , Cat# 8592S; RRID: AB_10891784.

Techniques: Infection, Western Blot

Journal: Immunity

Article Title: Flexible Usage and Interconnectivity of Diverse Cell Death Pathways Protect against Intracellular Infection

doi: 10.1016/j.immuni.2020.07.004

Figure Lengend Snippet: Caspase-11 Can Compensate for the Loss of Caspases-1 and -8 to Ensure GSDMD-Mediated Killing of Salmonella -Infected Cells (A) LDH release cell death assay of Salmonella SL1344 (MOI = 50) -infected iBMDMs of the indicated genotypes that had been left untreated or treated with VX-765 or Emricasan. Data pooled from two or more experiments. Mean and SEM are shown. ∗∗ p < 0.005, ns p > 0.05 = not significant. (B) LDH release cell death assays of iBMDMs of the indicated genotypes that had been infected with Salmonella SL1344 (MOI = 50). Data pooled from two or more experiments. Mean and SEM are shown. ∗∗ p < 0.005, ns p > 0.05 = not significant. (C) LDH release cell death assays of Salmonella -infected iBMDMs of the indicated genotypes or Casp1 –/– ;Casp8 –/– ;Ripk3 –/– iBMDMs that had been left untreated or treated with Emricasan and infected with Salmonella SL1344 (MOI = 50). Data pooled from two or more experiments. Mean and SEM are shown. ∗∗ p < 0.005, ns p > 0.05 = not significant. (D) Immunoblot analysis of caspase-11, GSDMD, BID, and PARP in Casp1 –/– ;Casp8 –/– ;Ripk3 –/– iBMDMs that had been left untreated or treated with Emricasan and infected with Salmonella SL1344 (MOI = 50). WT iBMDMs that had been left untreated or treated with LPS for 4 h were used as a control for the induction of caspase-11. Probing for β-actin served as a loading control. Please see also Figure S4 .

Article Snippet: rabbit anti-cleaved caspase-8 (D5B2) , CST , Cat# 8592S; RRID: AB_10891784.

Techniques: Infection, Western Blot

Journal: Immunity

Article Title: Flexible Usage and Interconnectivity of Diverse Cell Death Pathways Protect against Intracellular Infection

doi: 10.1016/j.immuni.2020.07.004

Figure Lengend Snippet: Caspase-1 Can Activate Caspases-3, -7, and -9 Independently of Caspase-8 and BID (A) LDH release cell death assays of iBMDMs of the indicated genotypes that had been infected with Salmonella SL1344 (MOI = 50). Data pooled from two or more experiments. Mean and SEM are shown. ∗∗ p < 0.005, ns p > 0.05 = not significant. (B) iBMDMs of the indicated genotypes were infected with Salmonella SL1344 (MOI = 50) and cleavage associated with activation of the indicated cell death proteins was analyzed by immunoblotting at the indicated time points. Probing for β-actin served as a loading control. (C) LDH release cell death assays of iBMDMs of the indicated genotypes that had been infected with Salmonella SL1344 (MOI = 50). Data pooled from two or more experiments. Mean and SEM are shown. ∗∗ p < 0.005, ns p > 0.05 = not significant. (D) GsdmD –/– ;Casp8 –/– ;Ripk3 –/– ;Mlkl –/– ;Casp3 –/– ;Casp7 –/– iBMDMs were infected with Salmonella SL1344 (MOI = 50) and expression and cleavage associated with activation of the indicated cell death proteins was analyzed by immunoblotting at the indicated time points. Probing for β-actin served as a loading control. (E) LDH release cell death assays of iBMDMs of the indicated genotypes that had been infected with Salmonella SL1344 (MOI = 50). Data pooled from two or more experiments. Mean and SEM are shown. ns p > 0.05 = not significant. (F) LDH release cell death assays of iBMDMs of the indicated genotypes that had been infected with Salmonella SL1344 (MOI = 50). Data pooled from two or more experiments. Mean and SEM are shown. ns p > 0.05 = not significant. Please see also Figure S5 .

Article Snippet: rabbit anti-cleaved caspase-8 (D5B2) , CST , Cat# 8592S; RRID: AB_10891784.

Techniques: Infection, Activation Assay, Western Blot, Expressing

Journal: Immunity

Article Title: Flexible Usage and Interconnectivity of Diverse Cell Death Pathways Protect against Intracellular Infection

doi: 10.1016/j.immuni.2020.07.004

Figure Lengend Snippet: CRISPR Screen Reveals a Central Role for Caspase-1 in Mediating Salmonella -Infection-Induced Cell Death Independent of All Known Downstream Effectors of Cell Killing (A) LDH release cell death assays of iBMDMs of the indicated genotypes that had been infected with Salmonella SL1344 (MOI = 50). Data pooled from two or more experiments. Mean and SEM are shown. ∗ p < 0.05, ns p > 0.05 = not significant. (B) GsdmD –/– ;Bid –/– ;Mlkl –/– ;Casp3 –/– ;Casp7 –/– ;Casp9 –/– iBMDM whole genome CRISPR-Cas9 screen mean-difference (MD) plot showing log-fold change versus average log counts per million (CPM) after three rounds of infection with Salmonella SL1344 (MOI = 50) (please also see A and S5B). (C) GsdmD –/– ;Bid –/– ;Mlkl –/– ;Casp3 –/– ;Casp7 –/– ;Casp9 –/– iBMDMs were infected with Salmonella SL1344 (MOI = 50) and cleavage associated with activation of caspases-1 and -8 was analyzed by immunoblotting at the indicated time points. Probing for β-actin served as a loading control. (D) LDH release cell death assays of WT, Casp1 –/– ;Casp11 –/– ;Casp12 –/– ;Casp8 –/– ;Ripk3 –/– , and two independent clones (#1 and #2) of GsdmD –/– ;Bid –/– ;Mlkl –/– ;Casp3 –/– ;Casp7 –/– ;Casp9 –/– ;Casp1 –/– iBMDMs that had been infected with Salmonella SL1344 (MOI = 50). Data pooled from two or more experiments. Mean and SEM are shown. ns p > 0.05 = not significant. Please see also Figure S6 .

Article Snippet: rabbit anti-cleaved caspase-8 (D5B2) , CST , Cat# 8592S; RRID: AB_10891784.

Techniques: CRISPR, Infection, Activation Assay, Western Blot, Clone Assay

Journal: Immunity

Article Title: Flexible Usage and Interconnectivity of Diverse Cell Death Pathways Protect against Intracellular Infection

doi: 10.1016/j.immuni.2020.07.004

Figure Lengend Snippet: Caspase-1 Can Act Upstream of and Requires Caspase-8 to Induce Cell Death in the Absence of All Known Downstream Effectors of Pyroptosis and Apoptosis (A) iBMDMs of the indicated genotypes were infected with Samonella SL1344 (MOI = 50) and cleavage associated with activation of caspases-1 and -8 was analyzed by immunoblotting at the indicated time points. Probing for HSP70 served as loading control. (B) LDH release death assays of Salmonella SL1344 (MOI = 50) -infected Casp1 –/– ;Casp11 –/– ;Casp12 –/– ;Casp8 –/– ;Ripk3 –/– and GsdmD –/– ;Bid –/– ;Mlkl –/– ;Casp3 –/– ;Casp7 –/– ;Casp9 –/– iBMDMs that had been left untreated or treated with VX-765 or Emricasan. Data pooled from two experiments. Mean and SEM are shown. ∗∗ p < 0.005, ns p > 0.05 = not significant. (C) Immunoblot analysis of caspases-1 and -8 activation at the indicated time points in Salmonella SL1344 (MOI = 50) -infected GsdmD –/– ;Bid –/– ;Mlkl –/– ;Casp3 –/– ;7 –/– ;9 –/– iBMDMs that had been left untreated or treated with VX-765. Probing for HSP70 served as a loading control. Please see also Figure S6 .

Article Snippet: rabbit anti-cleaved caspase-8 (D5B2) , CST , Cat# 8592S; RRID: AB_10891784.

Techniques: Infection, Activation Assay, Western Blot

Journal: Immunity

Article Title: Flexible Usage and Interconnectivity of Diverse Cell Death Pathways Protect against Intracellular Infection

doi: 10.1016/j.immuni.2020.07.004

Figure Lengend Snippet:

Article Snippet: rabbit anti-cleaved caspase-8 (D5B2) , CST , Cat# 8592S; RRID: AB_10891784.

Techniques: Recombinant, Protease Inhibitor, Western Blot, Lactate Dehydrogenase Assay, Plasmid Preparation, Sequencing, Software

Journal: Immunity

Article Title: Flexible Usage and Interconnectivity of Diverse Cell Death Pathways Protect against Intracellular Infection

doi: 10.1016/j.immuni.2020.07.004

Figure Lengend Snippet: Combined Loss of Caspases-1, -11, -12, and -8 (plus RIPK3) Leads to Lack of Bacterial Control upon Salmonella Infection (A) Bacterial replication over time in WT and Casp1 –/– ;Casp11 –/– mice infected with Salmonella Δ AroA (200 CFU). n = 10−22 mice per group per time point. Mean and SEM are shown. ∗∗ p < 0.005, ∗ p < 0.05, ns p > 0.05 = not significant. (B) Bacterial loads in spleen and liver of mice of the indicated genotypes 3 weeks post-infection with Salmonella Δ AroA (200 CFU). n = 7−48 mice per genotype. Mean and SEM are shown. ∗∗ p < 0.005, ∗ p < 0.05, ns p > 0.05 = not significant. (C) Bacterial loads in spleen and liver from mice of the indicated genotypes 1 to 3 weeks post-infection with Salmonella Δ AroA (200 CFU). n = 3−4 mice per genotype and time point. Mean and SEM are shown. ∗∗ p < 0.005, ∗ p < 0.05, ns p > 0.05 = not significant. (D) Mouse survival curves and corresponding bacterial loads in the spleen and liver at time of sacrifice in WT and Casp1 –/– ;Casp11 –/– ;Casp12 –/– ;Casp8 –/– ;Ripk3 −/− mice infected with Salmonella Δ AroA (200 CFU). n = 7−8 mice per genotype. Mean and SEM are shown. ∗∗ p < 0.005. (E) Bone marrow chimeras of the indicated genotypes were infected with Salmonella Δ AroA (200 CFU) and culled for analysis of bacterial loads in spleen and liver 3 weeks post-infection. n = 10 mice per group. Mean and SEM are shown. ∗∗ p < 0.005. Please also see Figure S1 .

Article Snippet: Proteins were transferred onto nitrocellulose membranes, blocked with either 5% skim milk (Devondale, Brunswick, Australia) or 5% BSA (for phospho-proteins) in PBS with 0.05% Tween-20 (PBST) for 1 h, and detected using the following primary antibodies: rat anti-caspase-11 (4E11, Enzo Life Sciences), rat anti-caspase-1 (1E11, Enzo Life Sciences), rat anti-MLKL (3H1; available from Merck), rabbit anti-phospho S345 MLKL (EPR9515[2]; Abcam, Cambridge, UK), rat anti-caspase-8 (3B10; Enzo Life Sciences), rabbit anti-cleaved caspase-8 (D5B2; Cell Signaling Technology), rabbit anti-RIPK3 (ProSci, Poway, CA, USA), rabbit anti-cleaved caspase-3 (Asp175; Cell Signaling Technology), rabbit anti-caspase-7 (D2Q3L, Cell Signaling), rabbit anti-caspase-9 (Cell Signaling Technology), rabbit anti-GSDMD (EPR19828, Abcam), mouse anti-PARP (C2-10, Santa Cruz), rat anti-BID (2D1-3, WEHI), mouse anti-HSP70 (BRM-22, Sigma Alrich) and rabbit anti-β-actin-HRP (Cell Signaling Technology).

Techniques: Infection

Journal: Immunity

Article Title: Flexible Usage and Interconnectivity of Diverse Cell Death Pathways Protect against Intracellular Infection

doi: 10.1016/j.immuni.2020.07.004

Figure Lengend Snippet: Caspase-8-Mediated Apoptosis Is the Default Backup Mechanism when Caspases-1- and 11-Mediated Pyroptosis Is Disabled in Salmonella -Infected iBMDMs (A) LDH release cell death assay of iBMDMs of the indicated genotypes after infection with Salmonella SL1344 (MOI = 50). Data pooled from two or more experiments. Mean and SEM are shown. ∗∗ p < 0.005, ns p > 0.05 = not significant. (B) Immunoblot analysis of the indicated proteins in iBMDMs of the indicated genotypes after infection with Salmonella SL1344 (MOI = 50). Probing for β-actin served as a loading control. (C) LDH release cell death assay of Salmonella SL1344 (MOI = 50) -infected WT and Casp1 –/– ;Casp11 –/– ;Casp12 –/– iBMDMs that had been left untreated or treated with the RIPK1 inhibitor, Nec1s (30 μM). Data pooled from two or more experiments. Mean and SEM are shown. ∗∗ p < 0.005, ns p > 0.05 = not significant. (D) Immunoblot analysis of the indicated proteins in Salmonella SL1344 (MOI = 50) -infected WT and Casp1 –/– ;Casp11 –/– ;Casp12 –/– iBMDMs that had been left untreated or treated with the RIPK1 inhibitor, Nec1s (30 μM). Probing for β-actin served as a loading control. Please see also and .

Article Snippet: Proteins were transferred onto nitrocellulose membranes, blocked with either 5% skim milk (Devondale, Brunswick, Australia) or 5% BSA (for phospho-proteins) in PBS with 0.05% Tween-20 (PBST) for 1 h, and detected using the following primary antibodies: rat anti-caspase-11 (4E11, Enzo Life Sciences), rat anti-caspase-1 (1E11, Enzo Life Sciences), rat anti-MLKL (3H1; available from Merck), rabbit anti-phospho S345 MLKL (EPR9515[2]; Abcam, Cambridge, UK), rat anti-caspase-8 (3B10; Enzo Life Sciences), rabbit anti-cleaved caspase-8 (D5B2; Cell Signaling Technology), rabbit anti-RIPK3 (ProSci, Poway, CA, USA), rabbit anti-cleaved caspase-3 (Asp175; Cell Signaling Technology), rabbit anti-caspase-7 (D2Q3L, Cell Signaling), rabbit anti-caspase-9 (Cell Signaling Technology), rabbit anti-GSDMD (EPR19828, Abcam), mouse anti-PARP (C2-10, Santa Cruz), rat anti-BID (2D1-3, WEHI), mouse anti-HSP70 (BRM-22, Sigma Alrich) and rabbit anti-β-actin-HRP (Cell Signaling Technology).

Techniques: Infection, Western Blot

Journal: Immunity

Article Title: Flexible Usage and Interconnectivity of Diverse Cell Death Pathways Protect against Intracellular Infection

doi: 10.1016/j.immuni.2020.07.004

Figure Lengend Snippet: Caspase-11 Can Compensate for the Loss of Caspases-1 and -8 to Ensure GSDMD-Mediated Killing of Salmonella -Infected Cells (A) LDH release cell death assay of Salmonella SL1344 (MOI = 50) -infected iBMDMs of the indicated genotypes that had been left untreated or treated with VX-765 or Emricasan. Data pooled from two or more experiments. Mean and SEM are shown. ∗∗ p < 0.005, ns p > 0.05 = not significant. (B) LDH release cell death assays of iBMDMs of the indicated genotypes that had been infected with Salmonella SL1344 (MOI = 50). Data pooled from two or more experiments. Mean and SEM are shown. ∗∗ p < 0.005, ns p > 0.05 = not significant. (C) LDH release cell death assays of Salmonella -infected iBMDMs of the indicated genotypes or Casp1 –/– ;Casp8 –/– ;Ripk3 –/– iBMDMs that had been left untreated or treated with Emricasan and infected with Salmonella SL1344 (MOI = 50). Data pooled from two or more experiments. Mean and SEM are shown. ∗∗ p < 0.005, ns p > 0.05 = not significant. (D) Immunoblot analysis of caspase-11, GSDMD, BID, and PARP in Casp1 –/– ;Casp8 –/– ;Ripk3 –/– iBMDMs that had been left untreated or treated with Emricasan and infected with Salmonella SL1344 (MOI = 50). WT iBMDMs that had been left untreated or treated with LPS for 4 h were used as a control for the induction of caspase-11. Probing for β-actin served as a loading control. Please see also Figure S4 .

Article Snippet: Proteins were transferred onto nitrocellulose membranes, blocked with either 5% skim milk (Devondale, Brunswick, Australia) or 5% BSA (for phospho-proteins) in PBS with 0.05% Tween-20 (PBST) for 1 h, and detected using the following primary antibodies: rat anti-caspase-11 (4E11, Enzo Life Sciences), rat anti-caspase-1 (1E11, Enzo Life Sciences), rat anti-MLKL (3H1; available from Merck), rabbit anti-phospho S345 MLKL (EPR9515[2]; Abcam, Cambridge, UK), rat anti-caspase-8 (3B10; Enzo Life Sciences), rabbit anti-cleaved caspase-8 (D5B2; Cell Signaling Technology), rabbit anti-RIPK3 (ProSci, Poway, CA, USA), rabbit anti-cleaved caspase-3 (Asp175; Cell Signaling Technology), rabbit anti-caspase-7 (D2Q3L, Cell Signaling), rabbit anti-caspase-9 (Cell Signaling Technology), rabbit anti-GSDMD (EPR19828, Abcam), mouse anti-PARP (C2-10, Santa Cruz), rat anti-BID (2D1-3, WEHI), mouse anti-HSP70 (BRM-22, Sigma Alrich) and rabbit anti-β-actin-HRP (Cell Signaling Technology).

Techniques: Infection, Western Blot

Journal: Immunity

Article Title: Flexible Usage and Interconnectivity of Diverse Cell Death Pathways Protect against Intracellular Infection

doi: 10.1016/j.immuni.2020.07.004

Figure Lengend Snippet: Caspase-1 Can Activate Caspases-3, -7, and -9 Independently of Caspase-8 and BID (A) LDH release cell death assays of iBMDMs of the indicated genotypes that had been infected with Salmonella SL1344 (MOI = 50). Data pooled from two or more experiments. Mean and SEM are shown. ∗∗ p < 0.005, ns p > 0.05 = not significant. (B) iBMDMs of the indicated genotypes were infected with Salmonella SL1344 (MOI = 50) and cleavage associated with activation of the indicated cell death proteins was analyzed by immunoblotting at the indicated time points. Probing for β-actin served as a loading control. (C) LDH release cell death assays of iBMDMs of the indicated genotypes that had been infected with Salmonella SL1344 (MOI = 50). Data pooled from two or more experiments. Mean and SEM are shown. ∗∗ p < 0.005, ns p > 0.05 = not significant. (D) GsdmD –/– ;Casp8 –/– ;Ripk3 –/– ;Mlkl –/– ;Casp3 –/– ;Casp7 –/– iBMDMs were infected with Salmonella SL1344 (MOI = 50) and expression and cleavage associated with activation of the indicated cell death proteins was analyzed by immunoblotting at the indicated time points. Probing for β-actin served as a loading control. (E) LDH release cell death assays of iBMDMs of the indicated genotypes that had been infected with Salmonella SL1344 (MOI = 50). Data pooled from two or more experiments. Mean and SEM are shown. ns p > 0.05 = not significant. (F) LDH release cell death assays of iBMDMs of the indicated genotypes that had been infected with Salmonella SL1344 (MOI = 50). Data pooled from two or more experiments. Mean and SEM are shown. ns p > 0.05 = not significant. Please see also Figure S5 .

Article Snippet: Proteins were transferred onto nitrocellulose membranes, blocked with either 5% skim milk (Devondale, Brunswick, Australia) or 5% BSA (for phospho-proteins) in PBS with 0.05% Tween-20 (PBST) for 1 h, and detected using the following primary antibodies: rat anti-caspase-11 (4E11, Enzo Life Sciences), rat anti-caspase-1 (1E11, Enzo Life Sciences), rat anti-MLKL (3H1; available from Merck), rabbit anti-phospho S345 MLKL (EPR9515[2]; Abcam, Cambridge, UK), rat anti-caspase-8 (3B10; Enzo Life Sciences), rabbit anti-cleaved caspase-8 (D5B2; Cell Signaling Technology), rabbit anti-RIPK3 (ProSci, Poway, CA, USA), rabbit anti-cleaved caspase-3 (Asp175; Cell Signaling Technology), rabbit anti-caspase-7 (D2Q3L, Cell Signaling), rabbit anti-caspase-9 (Cell Signaling Technology), rabbit anti-GSDMD (EPR19828, Abcam), mouse anti-PARP (C2-10, Santa Cruz), rat anti-BID (2D1-3, WEHI), mouse anti-HSP70 (BRM-22, Sigma Alrich) and rabbit anti-β-actin-HRP (Cell Signaling Technology).

Techniques: Infection, Activation Assay, Western Blot, Expressing

Journal: Immunity

Article Title: Flexible Usage and Interconnectivity of Diverse Cell Death Pathways Protect against Intracellular Infection

doi: 10.1016/j.immuni.2020.07.004

Figure Lengend Snippet: CRISPR Screen Reveals a Central Role for Caspase-1 in Mediating Salmonella -Infection-Induced Cell Death Independent of All Known Downstream Effectors of Cell Killing (A) LDH release cell death assays of iBMDMs of the indicated genotypes that had been infected with Salmonella SL1344 (MOI = 50). Data pooled from two or more experiments. Mean and SEM are shown. ∗ p < 0.05, ns p > 0.05 = not significant. (B) GsdmD –/– ;Bid –/– ;Mlkl –/– ;Casp3 –/– ;Casp7 –/– ;Casp9 –/– iBMDM whole genome CRISPR-Cas9 screen mean-difference (MD) plot showing log-fold change versus average log counts per million (CPM) after three rounds of infection with Salmonella SL1344 (MOI = 50) (please also see A and S5B). (C) GsdmD –/– ;Bid –/– ;Mlkl –/– ;Casp3 –/– ;Casp7 –/– ;Casp9 –/– iBMDMs were infected with Salmonella SL1344 (MOI = 50) and cleavage associated with activation of caspases-1 and -8 was analyzed by immunoblotting at the indicated time points. Probing for β-actin served as a loading control. (D) LDH release cell death assays of WT, Casp1 –/– ;Casp11 –/– ;Casp12 –/– ;Casp8 –/– ;Ripk3 –/– , and two independent clones (#1 and #2) of GsdmD –/– ;Bid –/– ;Mlkl –/– ;Casp3 –/– ;Casp7 –/– ;Casp9 –/– ;Casp1 –/– iBMDMs that had been infected with Salmonella SL1344 (MOI = 50). Data pooled from two or more experiments. Mean and SEM are shown. ns p > 0.05 = not significant. Please see also Figure S6 .

Article Snippet: Proteins were transferred onto nitrocellulose membranes, blocked with either 5% skim milk (Devondale, Brunswick, Australia) or 5% BSA (for phospho-proteins) in PBS with 0.05% Tween-20 (PBST) for 1 h, and detected using the following primary antibodies: rat anti-caspase-11 (4E11, Enzo Life Sciences), rat anti-caspase-1 (1E11, Enzo Life Sciences), rat anti-MLKL (3H1; available from Merck), rabbit anti-phospho S345 MLKL (EPR9515[2]; Abcam, Cambridge, UK), rat anti-caspase-8 (3B10; Enzo Life Sciences), rabbit anti-cleaved caspase-8 (D5B2; Cell Signaling Technology), rabbit anti-RIPK3 (ProSci, Poway, CA, USA), rabbit anti-cleaved caspase-3 (Asp175; Cell Signaling Technology), rabbit anti-caspase-7 (D2Q3L, Cell Signaling), rabbit anti-caspase-9 (Cell Signaling Technology), rabbit anti-GSDMD (EPR19828, Abcam), mouse anti-PARP (C2-10, Santa Cruz), rat anti-BID (2D1-3, WEHI), mouse anti-HSP70 (BRM-22, Sigma Alrich) and rabbit anti-β-actin-HRP (Cell Signaling Technology).

Techniques: CRISPR, Infection, Activation Assay, Western Blot, Clone Assay

Journal: Immunity

Article Title: Flexible Usage and Interconnectivity of Diverse Cell Death Pathways Protect against Intracellular Infection

doi: 10.1016/j.immuni.2020.07.004

Figure Lengend Snippet: Caspase-1 Can Act Upstream of and Requires Caspase-8 to Induce Cell Death in the Absence of All Known Downstream Effectors of Pyroptosis and Apoptosis (A) iBMDMs of the indicated genotypes were infected with Samonella SL1344 (MOI = 50) and cleavage associated with activation of caspases-1 and -8 was analyzed by immunoblotting at the indicated time points. Probing for HSP70 served as loading control. (B) LDH release death assays of Salmonella SL1344 (MOI = 50) -infected Casp1 –/– ;Casp11 –/– ;Casp12 –/– ;Casp8 –/– ;Ripk3 –/– and GsdmD –/– ;Bid –/– ;Mlkl –/– ;Casp3 –/– ;Casp7 –/– ;Casp9 –/– iBMDMs that had been left untreated or treated with VX-765 or Emricasan. Data pooled from two experiments. Mean and SEM are shown. ∗∗ p < 0.005, ns p > 0.05 = not significant. (C) Immunoblot analysis of caspases-1 and -8 activation at the indicated time points in Salmonella SL1344 (MOI = 50) -infected GsdmD –/– ;Bid –/– ;Mlkl –/– ;Casp3 –/– ;7 –/– ;9 –/– iBMDMs that had been left untreated or treated with VX-765. Probing for HSP70 served as a loading control. Please see also Figure S6 .

Article Snippet: Proteins were transferred onto nitrocellulose membranes, blocked with either 5% skim milk (Devondale, Brunswick, Australia) or 5% BSA (for phospho-proteins) in PBS with 0.05% Tween-20 (PBST) for 1 h, and detected using the following primary antibodies: rat anti-caspase-11 (4E11, Enzo Life Sciences), rat anti-caspase-1 (1E11, Enzo Life Sciences), rat anti-MLKL (3H1; available from Merck), rabbit anti-phospho S345 MLKL (EPR9515[2]; Abcam, Cambridge, UK), rat anti-caspase-8 (3B10; Enzo Life Sciences), rabbit anti-cleaved caspase-8 (D5B2; Cell Signaling Technology), rabbit anti-RIPK3 (ProSci, Poway, CA, USA), rabbit anti-cleaved caspase-3 (Asp175; Cell Signaling Technology), rabbit anti-caspase-7 (D2Q3L, Cell Signaling), rabbit anti-caspase-9 (Cell Signaling Technology), rabbit anti-GSDMD (EPR19828, Abcam), mouse anti-PARP (C2-10, Santa Cruz), rat anti-BID (2D1-3, WEHI), mouse anti-HSP70 (BRM-22, Sigma Alrich) and rabbit anti-β-actin-HRP (Cell Signaling Technology).

Techniques: Infection, Activation Assay, Western Blot

Journal: Immunity

Article Title: Flexible Usage and Interconnectivity of Diverse Cell Death Pathways Protect against Intracellular Infection

doi: 10.1016/j.immuni.2020.07.004

Figure Lengend Snippet:

Article Snippet: Proteins were transferred onto nitrocellulose membranes, blocked with either 5% skim milk (Devondale, Brunswick, Australia) or 5% BSA (for phospho-proteins) in PBS with 0.05% Tween-20 (PBST) for 1 h, and detected using the following primary antibodies: rat anti-caspase-11 (4E11, Enzo Life Sciences), rat anti-caspase-1 (1E11, Enzo Life Sciences), rat anti-MLKL (3H1; available from Merck), rabbit anti-phospho S345 MLKL (EPR9515[2]; Abcam, Cambridge, UK), rat anti-caspase-8 (3B10; Enzo Life Sciences), rabbit anti-cleaved caspase-8 (D5B2; Cell Signaling Technology), rabbit anti-RIPK3 (ProSci, Poway, CA, USA), rabbit anti-cleaved caspase-3 (Asp175; Cell Signaling Technology), rabbit anti-caspase-7 (D2Q3L, Cell Signaling), rabbit anti-caspase-9 (Cell Signaling Technology), rabbit anti-GSDMD (EPR19828, Abcam), mouse anti-PARP (C2-10, Santa Cruz), rat anti-BID (2D1-3, WEHI), mouse anti-HSP70 (BRM-22, Sigma Alrich) and rabbit anti-β-actin-HRP (Cell Signaling Technology).

Techniques: Recombinant, Protease Inhibitor, Western Blot, Lactate Dehydrogenase Assay, Plasmid Preparation, Sequencing, Software