Journal: Biochemical Journal

Article Title: The VEGFR/PDGFR tyrosine kinase inhibitor, ABT-869, blocks necroptosis by targeting RIPK1 kinase

doi: 10.1042/BCJ20230035

Figure Lengend Snippet: ( A ) Schematic of the necroptosis pathway. TNF (T) activates TNFR1, the Smac-mimetic Compound A (S) blocks cIAP activity and the pan-caspase inhibitor Q-VD-OPh (Q) blocks caspase-8 activity. This TSQ stimulus results in activation of RIPK1 and RIPK3, and subsequent phosphorylation and activation of MLKL, which causes MLKL-mediated membrane disruption and cell death. ( B ) Schematic of the constitutively activated mouse MLKL mutant, Q343A. Expression of MLKL Q343A using doxycycline causes cell death in the absence of upstream necroptotic stimuli. This enabled a cell-based phenotypic screen for small molecules that modulate necroptosis at the level or downstream of MLKL activation. The skull and crossbones image (Mycomorphbox_Deadly.png; by Sven Manguard) in ( A , B ) was used under a Creative Commons Attribution-Share Alike 4.0 license. ( C ) Schematic of the cell-based phenotypic screen. A total of 5632 compounds from the WEHI small molecule library along with 40 kinase inhibitors were screened against wild-type or Mlkl −/− mouse dermal fibroblast (MDF) cells expressing the MLKL Q343A mutant. The ability of the small molecules to inhibit cell death was measured by CellTiter-Glo cell viability assays. ABT-869, a VEGF and PDGF receptor tyrosine kinase inhibitor, was identified as a hit. See also Supplementary Figure S1A. ( D ) Chemical structure of ABT-869 and its analogue WEHI-615. ( E ) Wild-type mouse dermal fibroblast (MDF) cells expressing the doxycycline-inducible MLKL Q343A mutant to trigger constitutive necroptosis were treated with DMSO alone, doxycycline (Dox; 1 µg/ml) and DMSO, or Dox and ABT-869 (1 µM). Cell viability was quantified by CellTiter-Glo. Data represent the mean of ≥2 technical replicates from a single experiment, with individual data points shown. See also Supplementary Figure S1A. ( F ) Mlkl −/− mouse dermal fibroblast (MDF) cells expressing the doxycycline-inducible MLKL Q343A mutant to trigger constitutive necroptosis were treated with DMSO alone, doxycycline (Dox; 1 µg/ml) and DMSO, or Dox and ABT-869 (1 µM). Cell viability was quantified by CellTiter-Glo. Data represent the mean of ≥2 technical replicates from a single experiment, with individual data points shown. See also Supplementary Figure S1A. ( G ) Wild-type mouse dermal fibroblast (MDF) cells were stimulated with TSQ (TNF, Smac-mimetic, Q-VD-OPh) to induce necroptosis and treated with increasing concentrations of ABT-869 or WEHI-615. Cell death was quantified by propidium iodide (PI) staining using flow cytometry. Data represent the mean of n = 4 independent experiments and errors bars represent SEM.

Article Snippet: Phospho-mouse RIPK3 , Rabbit anti-mouse RIPK3 pT231/pS232 , Kindly supplied by Genentech, GEN135-35-9 [ ] , N/A , 1 : 5000.

Techniques: Activity Assay, Activation Assay, Mutagenesis, Expressing, Staining, Flow Cytometry

Journal: Biochemical Journal

Article Title: The VEGFR/PDGFR tyrosine kinase inhibitor, ABT-869, blocks necroptosis by targeting RIPK1 kinase

doi: 10.1042/BCJ20230035

Figure Lengend Snippet: ( A , B ) Wild-type mouse dermal fibroblast (MDF) cells were treated with increasing concentrations of ABT-869 or control compounds, RIPK3 inhibitors GSK′872 and GSK′843, DMSO alone or left untreated (UT) for 1 h then stimulated with TSQ (TNF, Smac-mimetic, Q-VD-OPh) ( A ) or TSZ (TNF, Smac-mimetic, z-VAD-fmk) ( B ) for 24 h to induce necroptosis. Cell death was quantified by propidium iodide (PI) staining using flow cytometry. Data represent the mean of n = 3 ( A ) or n = 4 ( B ) independent experiments and error bars represent SEM. ( C – F ) Human U937 cells were treated with increasing concentrations of ABT-869 or control compounds, MLKL inhibitor NSA and RIPK1 inhibitor GSK′481, DMSO alone or left untreated (UT) for 1 h then stimulated with TSQ (TNF, Smac-mimetic, Q-VD-OPh) for 48 h ( C ) or TSI (TNF, Smac-mimetic, IDN-6556) for 24 h ( E ) to induce necroptosis. Parallel experiments were performed to assess protection of TSQ ( D ) or TSI ( F ) induced death in the presence of the ABT-869 analogue, WEHI-615. Cell death was monitored by SPY505 (live cells) and propidium iodide (PI; dead cells) uptake using IncuCyte live cell imaging. One representative result shown from n = 4 ( C , D ) or n = 3 ( E , F ) independent experiments. See also Supplementary Figure S2A–H.

Article Snippet: Phospho-mouse RIPK3 , Rabbit anti-mouse RIPK3 pT231/pS232 , Kindly supplied by Genentech, GEN135-35-9 [ ] , N/A , 1 : 5000.

Techniques: Staining, Flow Cytometry, Live Cell Imaging

Journal: Biochemical Journal

Article Title: The VEGFR/PDGFR tyrosine kinase inhibitor, ABT-869, blocks necroptosis by targeting RIPK1 kinase

doi: 10.1042/BCJ20230035

Figure Lengend Snippet: ( A ) Binding affinities ( K D ) of ABT-869 and WEHI-615 for human full-length MLKL, RIPK1 kinase domain and RIPK3 kinase domain measured by competition binding assays from the DiscoverX KINOME scan platform using the Kd ELECT service. Each value is the mean of two replicates. ( B – D ) Cellular Thermal Shift Assays (CETSA) in mouse and human cells. Mlkl −/− mouse dermal fibroblast (MDF) cells expressing MLKL Q343A ( B ), wild-type MDF cells ( C ) and human U937 cells ( D ) were treated with DMSO, ABT-869, WEHI-615, RIPK1 inhibitor Nec-1s, RIPK3 inhibitor GSK′872 or human RIPK1 inhibitor GSK′481 (all 20 µM). Cells were subjected to an increasing temperature gradient focused around the melting temperature of the protein of interest. Following the separation of soluble and insoluble proteins, the remaining soluble proteins were detected by Western blot. Red asterisks denote protein standards. One representative result shown from n = 3 ( B , C ) or n = 2–3 ( D ) independent experiments. See also Supplementary Figure S3A–C.

Article Snippet: Phospho-mouse RIPK3 , Rabbit anti-mouse RIPK3 pT231/pS232 , Kindly supplied by Genentech, GEN135-35-9 [ ] , N/A , 1 : 5000.

Techniques: Binding Assay, Expressing, Western Blot

Journal: Biochemical Journal

Article Title: The VEGFR/PDGFR tyrosine kinase inhibitor, ABT-869, blocks necroptosis by targeting RIPK1 kinase

doi: 10.1042/BCJ20230035

Figure Lengend Snippet: Thermal Shift Assays (TSA) with mouse and human RIPK1 and RIPK3 kinase domains. Increasing concentrations of ABT-869 or WEHI-615 were tested for their ability to alter the melting temperature ( T M ) of mouse RIPK1 (9.5 µg) ( A , B ), human RIPK1 (12 µg) ( C , D ), mouse RIPK3 (10 µg) ( E , F ) and human RIPK3 (6.5 µg) ( G , H ) compared with the positive controls Compound 2 for mouse RIPK1, GSK′481 for human RIPK1 and GSK′872 for mouse and human RIPK3 (all 30 µM). Data represent the mean of n = 3 independent experiments and error bars represent SEM. See also Supplementary Figure S4A–H.

Article Snippet: Phospho-mouse RIPK3 , Rabbit anti-mouse RIPK3 pT231/pS232 , Kindly supplied by Genentech, GEN135-35-9 [ ] , N/A , 1 : 5000.

Techniques:

Journal: Biochemical Journal

Article Title: The VEGFR/PDGFR tyrosine kinase inhibitor, ABT-869, blocks necroptosis by targeting RIPK1 kinase

doi: 10.1042/BCJ20230035

Figure Lengend Snippet: ( A – H ) In vitro phosphorylation assays with mouse and human RIPK1 and RIPK3 kinase domains measured by ADP-Glo Kinase Assays. Increasing concentrations of ABT-869 or WEHI-615 were tested for their ability to inhibit the autophosphorylation (IC 50 ) of mouse RIPK1 (200 nM) ( A , B ), human RIPK1 (200 nM) ( C , D ), mouse RIPK3 (10 nM) ( E , F ) and human RIPK3 (10 nM) ( G , H ). Data represent the mean of n = 3 ( A , B , E , F ) or n = 2 ( C , D , G , H ) independent experiments and error bars represent SEM. ( I ) Cellular phosphorylation assays. Wild-type mouse dermal fibroblast (MDF) cells were treated with DMSO, ABT-869, WEHI-615, RIPK1 inhibitor Nec-1s or RIPK3 inhibitor GSK′872 for 2 h then stimulated with TSI (TNF, Smac-mimetic, IDN-6556) for 2 h to induce autophosphorylation of RIPK1 and RIPK3. Ripk1 −/− Mlkl −/− MDF cells and Ripk3 −/− MDF cells were included as controls. Phospho-RIPK1 and phospho-RIPK3 protein levels were detected from whole cell lysates by Western blot. Red asterisks denote protein standards. One representative result shown from n = 3 independent experiments. See also Supplementary Figure S5A–C.

Article Snippet: Phospho-mouse RIPK3 , Rabbit anti-mouse RIPK3 pT231/pS232 , Kindly supplied by Genentech, GEN135-35-9 [ ] , N/A , 1 : 5000.

Techniques: In Vitro, Western Blot

Journal: Biochemical Journal

Article Title: The VEGFR/PDGFR tyrosine kinase inhibitor, ABT-869, blocks necroptosis by targeting RIPK1 kinase

doi: 10.1042/BCJ20230035

Figure Lengend Snippet:

Article Snippet: Phospho-mouse RIPK3 , Rabbit anti-mouse RIPK3 pT231/pS232 , Kindly supplied by Genentech, GEN135-35-9 [ ] , N/A , 1 : 5000.

Techniques: Western Blot, Produced, Transduction

Journal: Biochemical Journal

Article Title: The VEGFR/PDGFR tyrosine kinase inhibitor, ABT-869, blocks necroptosis by targeting RIPK1 kinase

doi: 10.1042/BCJ20230035

Figure Lengend Snippet:

Article Snippet: Phospho-mouse RIPK3 , Rabbit anti-mouse RIPK3 pT231/pS232 , Kindly supplied by Genentech, GEN135-35-9 [ ] , N/A , 1 : 5000.

Techniques:

Journal: Science Advances

Article Title: Caspase-8 inactivation drives autophagy-dependent inflammasome activation in myeloid cells

doi: 10.1126/sciadv.abn9912

Figure Lengend Snippet: ( A ) Expression of FADD and RIPK3 in Fadd +/+ Ripk3 +/+ , Fadd +/− Ripk3 −/− , and Fadd −/− Ripk3 −/− BMDMs, as determined by Western blot. ( B ) IETD induces IL-1β production in Fadd −/− Ripk3 −/− BMDCs stimulated with dectin-1 agonists. BMDCs were stimulated with curdlan (20 μg/ml) or Dzymosan (50 μg/ml), in the absence or presence of IETD (20 μM) for 8 hours, before analyzing IL-1β levels in culture supernatant by ELISA. Data are means ± SD of technical triplicates from one experiment and are representative of three independent experiments. ( C ) IETD + TLR agonists induce IL-1β production in Fadd −/− Ripk3 −/− BMDCs. BMDCs were stimulated with LPS (0.1 μg/ml) or R848 (1 μg/ml) in the presence or absence of IETD (20 μM) for 20 hours, and IL-1β levels were then determined. Data are means of three independent experiments. ( D and E ) IETD + TLR agonists induce IL-1β and IL-18 production in BMDMs. BMDMs were stimulated with LPS (0.1 μg/ml), R848 (1 μg/ml), or poly(I:C) (50 μg/ml) in the presence or absence of IETD (10 μM) for 20 hours, and then IL-1β (D) and IL-18 (E) production was determined. Data are means of three independent experiments. ( F ) IETD does not induce IL-1β production in TNF-stimulated Fadd −/− Ripk3 −/− BMDCs. BMDCs were stimulated with LPS (0.1 μg/ml) or TNF (50 ng/ml) in the presence or absence of IETD for 8 hours, and then IL-1β production was quantitated. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001 for two-way ANOVA followed by Si’ak’s multiple comparisons test.

Article Snippet: Anti-mouse RIPK3 (95702, RRID:AB_2721823), anti-pIKK (2078, RRID:AB_2079379), anti-IKKβ (8943, RRID:AB_11024092), anti-pJNK (9251, RRID:AB_331659), anti-JNK (9252, RRID:AB_2250373), anti-pp38 (9211, RRID:AB_331641), anti-p38 (9218, RRID:AB_10694846), anti-pERK (9101, RRID:AB_331646), anti-ERK (9102, RRID:AB_330744), anti-Atg5 (12994, RRID:AB_2630393), anti–Beclin-1 (3495, RRID:AB_1903911), anti–caspase-3 (9662, RRID:AB_331439), anti–cleaved caspase-3 (9661, RRID:AB_2341188), anti-mouse caspase-8 (4927, RRID:AB_2068301), and anti–cleaved caspase-8 (9429, RRID:AB_2068300) were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Expressing, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Science Advances

Article Title: Caspase-8 inactivation drives autophagy-dependent inflammasome activation in myeloid cells

doi: 10.1126/sciadv.abn9912

Figure Lengend Snippet: ( A ) Fadd −/− Ripk3 −/− BMDMs were stimulated with LPS (0.1 μg/ml) plus IETD (10 μM) for 4 hours, and levels of NLRP3, procaspase-1, ASC, and pro–IL-1β in cell lysates were then determined by immunoblotting. ( B and C ) Attenuated TLR activation signals in Fadd −/− Ripk3 −/− BMDMs. Fadd −/− Ripk3 −/− BMDMs were stimulated with LPS (0.1 μg/ml) for the indicated time frames. Cell lysates were then analyzed for p-IKK, IKKβ, and IκBα (B) and p-JNK, JNK, p-p38, p38, p-ERK, and ERK (C). ( D ) Formation of ASC pyroptosomes. BMDMs were stimulated with R848 (1 μg/ml) plus nigericin (N; 15 μM) or R848 (1 μg/ml) plus IETD (I; 10 μM). Total cell lysates were cross-linked by dextran sulfate sodium (DSS) and immunoblotted with anti-ASC. ( E ) BMDCs were stimulated with LPS (0.1 μg/ml) plus IETD (10 μM) for 6 hours (L + I) or LPS for 2 hours followed by nigericin (15 μM) for 20 min (L + N). Culture supernatants (SUP) were precipitated, and whole-cell lysates (WCL) were collected and analyzed for cleavage of procaspase-1 and pro–IL-1β. ( F ) BMDMs were stimulated with LPS (0.1 μg/ml) plus IETD (10 μM) for 10 hours (L + I) or LPS for 2 hours followed by nigericin (15 μM) for 20 min (L + N). Culture supernatants (SUP) were precipitated, and whole-cell lysates (WCL) were collected and analyzed for cleavage of procaspase-1, pro–IL-18, and GSDMD. Results are representative of two (B to D) or three (A, E, and F) independent experiments.

Article Snippet: Anti-mouse RIPK3 (95702, RRID:AB_2721823), anti-pIKK (2078, RRID:AB_2079379), anti-IKKβ (8943, RRID:AB_11024092), anti-pJNK (9251, RRID:AB_331659), anti-JNK (9252, RRID:AB_2250373), anti-pp38 (9211, RRID:AB_331641), anti-p38 (9218, RRID:AB_10694846), anti-pERK (9101, RRID:AB_331646), anti-ERK (9102, RRID:AB_330744), anti-Atg5 (12994, RRID:AB_2630393), anti–Beclin-1 (3495, RRID:AB_1903911), anti–caspase-3 (9662, RRID:AB_331439), anti–cleaved caspase-3 (9661, RRID:AB_2341188), anti-mouse caspase-8 (4927, RRID:AB_2068301), and anti–cleaved caspase-8 (9429, RRID:AB_2068300) were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Western Blot, Activation Assay

Journal: Science Advances

Article Title: Caspase-8 inactivation drives autophagy-dependent inflammasome activation in myeloid cells

doi: 10.1126/sciadv.abn9912

Figure Lengend Snippet: ( A ) BMDCs were treated with LPS plus IETD for 6 hours (L + I) or LPS for 2 hours followed by nigericin for 20 min (L + N). Culture supernatants and whole-cell lysates were collected and analyzed for cleavage of GSDMD. Short exp, 1-min exposure; Long exp, 3-min exposure. ( B and C ) Fadd −/− Ripk3 −/− BMDMs (B) and BMDCs (C) were treated with LPS (0.1 μg/ml) + IETD (10 μM) in the absence or presence of disulfiram (2.5 μM) for 16 hours before determining IL-1β (B and C) and IL-18 (B) production. ( D ) GSDMD, RIPK3, and FADD contents in Fadd +/− Ripk3 +/+ Gsdmd +/− , Fadd −/− Ripk3 −/− Gsdmd −/− , Fadd −/− Ripk3 −/− Gsdmd +/+ , and Fadd −/− Ripk3 −/− Gsdmd −/− BMDCs. ( E ) IL-1β production by Fadd +/− Ripk3 +/+ Gsdmd +/− , Fadd −/− Ripk3 −/− Gsdmd −/− , Fadd −/− Ripk3 −/− Gsdmd +/+ , and Fadd −/− Ripk3 −/− Gsdmd −/− BMDCs treated with LPS + IETD for 16 hours. ( F ) IL-1β production by Fadd +/− Ripk3 +/+ Gsdmd +/− , Fadd −/− Ripk3 −/− Gsdmd −/− , Fadd −/− Ripk3 −/− Gsdmd +/+ , and Fadd −/− Ripk3 −/− Gsdmd −/− BMDCs treated with LPS (0.1 μg/ml) for 3 hours followed by nigericin (20 μM) for 1 hour. Data (B, C, E, and F) are means of three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001 for two-way ANOVA followed by Sidak’s multiple comparisons test.

Article Snippet: Anti-mouse RIPK3 (95702, RRID:AB_2721823), anti-pIKK (2078, RRID:AB_2079379), anti-IKKβ (8943, RRID:AB_11024092), anti-pJNK (9251, RRID:AB_331659), anti-JNK (9252, RRID:AB_2250373), anti-pp38 (9211, RRID:AB_331641), anti-p38 (9218, RRID:AB_10694846), anti-pERK (9101, RRID:AB_331646), anti-ERK (9102, RRID:AB_330744), anti-Atg5 (12994, RRID:AB_2630393), anti–Beclin-1 (3495, RRID:AB_1903911), anti–caspase-3 (9662, RRID:AB_331439), anti–cleaved caspase-3 (9661, RRID:AB_2341188), anti-mouse caspase-8 (4927, RRID:AB_2068301), and anti–cleaved caspase-8 (9429, RRID:AB_2068300) were purchased from Cell Signaling Technology (Danvers, MA).

Techniques:

Journal: Science Advances

Article Title: Caspase-8 inactivation drives autophagy-dependent inflammasome activation in myeloid cells

doi: 10.1126/sciadv.abn9912

Figure Lengend Snippet: ( A ) BMDMs were stimulated with LPS (0.1 μg/ml) or poly(I:C) (50 μg/ml) plus IETD (10 μM) for 20 hours. LDH release and cell viability (determined by ATP assay) were analyzed. ( B ) BMDMs were stimulated with LPS plus IETD or zVAD (40 μM) for 16 hours, and cell viability was analyzed. ( C and D ) BMDMs were stimulated with LPS with or without IETD, in the absence (C) or presence of PI (D), for 16 hours. Scale bars, 25 μm (C) and 50 μm (D). Bright-field images are representative of three independent experiments. ( E ) Fadd −/− Ripk3 −/− BMDCs were transfected with control or caspase-8 siRNA for 24 hours, followed by LPS + IETD treatment for 8 hours, and LDH release was determined. ( F and G ) LDH release from BMDCs of indicated Gsdmd genotypes upon treatment with LPS + nigericin for 20 min (F) or with LPS + IETD for 8 hours (G). ( H ) Fadd −/− Ripk3 −/− BMDCs and BMDMs were stimulated with LPS + IETD without or with YVAD (20 μM) or Nec-1 (40 μM) for 18 hours, and then LDH release was determined. ( I ) BMDCs of indicated Casp1/11 genotypes were treated with LPS or LPS + IETD for 7.5 hours, and LDH release was quantified. Data represent four (A), five (E), and three (F to H) independent experiments, and mean values are indicated. For (B) and (I), data represent means ± SD of technical triplicates of an experiment representative of three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001 for two-way ANOVA followed by Sidak’s multiple comparisons test. ns, not significant.

Article Snippet: Anti-mouse RIPK3 (95702, RRID:AB_2721823), anti-pIKK (2078, RRID:AB_2079379), anti-IKKβ (8943, RRID:AB_11024092), anti-pJNK (9251, RRID:AB_331659), anti-JNK (9252, RRID:AB_2250373), anti-pp38 (9211, RRID:AB_331641), anti-p38 (9218, RRID:AB_10694846), anti-pERK (9101, RRID:AB_331646), anti-ERK (9102, RRID:AB_330744), anti-Atg5 (12994, RRID:AB_2630393), anti–Beclin-1 (3495, RRID:AB_1903911), anti–caspase-3 (9662, RRID:AB_331439), anti–cleaved caspase-3 (9661, RRID:AB_2341188), anti-mouse caspase-8 (4927, RRID:AB_2068301), and anti–cleaved caspase-8 (9429, RRID:AB_2068300) were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: ATP Assay, Transfection

Journal: Science Advances

Article Title: Caspase-8 inactivation drives autophagy-dependent inflammasome activation in myeloid cells

doi: 10.1126/sciadv.abn9912

Figure Lengend Snippet: ( A ) Age- and sex-matched Fadd +/− Ripk3 −/− and Fadd −/− Ripk3 −/− mice were intraperitoneally injected with IETD (5 mg/kg), and then survival was determined at the indicated time points. The P value was determined by a log-rank (Mantel-Cox) test. ( B ) Serum concentration of IL-6 in mice before and 3 hours after IETD administration. n = 3 (control) and n = 6 (IETD). ( C ) Peritoneal cells were isolated from mice 3 hours after PBS or IETD administration. Viability was assessed by means of PI staining and quantitated by flow cytometry. Data are mean values for the PBS group ( n = 3) or IETD group ( n = 6). ( D ) Peritoneal cells were isolated from mice 3 hours after PBS or IETD administration, before determining levels of IL-6 and IL-1β. Data are mean values from n = 3 (PBS) and n = 6 (IETD). ( E ) Fadd +/− Ripk3 −/− Nlrp3 −/− and Fadd −/− Ripk3 −/− Nlrp3 −/− mice were administered with IETD (5 mg/kg) before determining survival ( n = 7 for each group). The P value was determined by a log-rank (Mantel Cox) test. ( F ) Peritoneal cells were isolated from mice 3 hours after PBS or IETD administration, and then their production of IL-6 was determined ( n = 3 for each group). ( G ) Emricasan (20 mg/kg) was administered to Fadd +/− Ripk3 −/− ( n = 7) and Fadd −/− Ripk3 −/− ( n = 6) mice, before determining mouse survival. The P value was determined by a log-rank (Mantel-Cox) test. (B to D and F) * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001 for two-way ANOVA followed by a Sidak’s multiple comparison test.

Article Snippet: Anti-mouse RIPK3 (95702, RRID:AB_2721823), anti-pIKK (2078, RRID:AB_2079379), anti-IKKβ (8943, RRID:AB_11024092), anti-pJNK (9251, RRID:AB_331659), anti-JNK (9252, RRID:AB_2250373), anti-pp38 (9211, RRID:AB_331641), anti-p38 (9218, RRID:AB_10694846), anti-pERK (9101, RRID:AB_331646), anti-ERK (9102, RRID:AB_330744), anti-Atg5 (12994, RRID:AB_2630393), anti–Beclin-1 (3495, RRID:AB_1903911), anti–caspase-3 (9662, RRID:AB_331439), anti–cleaved caspase-3 (9661, RRID:AB_2341188), anti-mouse caspase-8 (4927, RRID:AB_2068301), and anti–cleaved caspase-8 (9429, RRID:AB_2068300) were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Injection, Concentration Assay, Isolation, Staining, Flow Cytometry

Journal: Science Advances

Article Title: Caspase-8 inactivation drives autophagy-dependent inflammasome activation in myeloid cells

doi: 10.1126/sciadv.abn9912

Figure Lengend Snippet: ( A ) Bright-field and PI staining images of BMDMs stimulated by LPS + IETD in the presence of 3-MA. Fadd −/− Ripk3 −/− BMDMs were treated with LPS (0.1 μg/ml) + IETD (10 μM) in the absence or presence of 3-MA (1 mM) for 18 hours. Images are representative of three independent experiments. Scale bar, 50 μm. ( B ) 3-MA inhibits IETD-induced cell death in Fadd −/− Ripk3 −/− myeloid cells. Fadd −/− Ripk3 −/− BMDCs and BMDMs were stimulated with LPS + IETD in the absence or presence of 3-MA (1 mM) for 8 hours (BMDCs) or 18 hours (BMDMs), and then LDH release was determined. LDH released from Ctrl cells treated with LPS + IETD was set as 1. Data represent mean values of four independent experiments. ( C ) Immunofluorescence analysis of DAPGreen in Fadd −/− Ripk3 −/− BMDMs. BMDMs were stained with DAPGreen (0.2 μM) for 30 min and then stimulated with LPS (0.1 μg/ml) plus IETD (10 μM) for 16 hours. Cell nuclei were stained with Hoechst 33342 Ready Flow reagent. Scale bar, 25 μm. Images are representative of four independent experiments. Average DAPGreen fluorescence intensity in a cell was quantified in ImageJ. The fluorescence intensity induced by LPS + IETD was compared to that of LPS alone. ( D ) 3-MA inhibits IETD-induced autophagy in Fadd −/− Ripk3 −/− BMDMs. Fadd −/− Ripk3 −/− BMDMs were stained with DAPGreen and stimulated with LPS + IETD as in (C), in the absence or presence of 3-MA (1 mM) for 16 hours. Images are representative of four independent experiments. Scale bar, 50 μm. Images were quantified as in (C). * P < 0.05, ** P < 0.01, and **** P < 0.0001 for two-way ANOVA followed by Sidak’s (B) or Tukey’s (C and D) multiple comparisons test.

Article Snippet: Anti-mouse RIPK3 (95702, RRID:AB_2721823), anti-pIKK (2078, RRID:AB_2079379), anti-IKKβ (8943, RRID:AB_11024092), anti-pJNK (9251, RRID:AB_331659), anti-JNK (9252, RRID:AB_2250373), anti-pp38 (9211, RRID:AB_331641), anti-p38 (9218, RRID:AB_10694846), anti-pERK (9101, RRID:AB_331646), anti-ERK (9102, RRID:AB_330744), anti-Atg5 (12994, RRID:AB_2630393), anti–Beclin-1 (3495, RRID:AB_1903911), anti–caspase-3 (9662, RRID:AB_331439), anti–cleaved caspase-3 (9661, RRID:AB_2341188), anti-mouse caspase-8 (4927, RRID:AB_2068301), and anti–cleaved caspase-8 (9429, RRID:AB_2068300) were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Staining, Immunofluorescence, Fluorescence

Journal: Science Advances

Article Title: Caspase-8 inactivation drives autophagy-dependent inflammasome activation in myeloid cells

doi: 10.1126/sciadv.abn9912

Figure Lengend Snippet: ( A to C ) Fadd −/− Ripk3 −/− BMDMs (A) and BMDCs (B and C) were stimulated with LPS (0.1 μg/ml) plus IETD (10 μM) in the absence (Ctrl) or presence of 3-MA (1 mM), chloroquine (10 μM), or NH 4 Cl (2 mM) for 18 hours (A) or 8 hours (B and C); then IL-1β (A, B) and IL-18 (A) levels were determined; and LDH release (C) was quantitated. ( D and E ) Fadd −/− Ripk3 −/− BMDCs were treated with LPS, IETD, or 3-MA as indicated. Culture supernatants (D) and whole-cell lysates (D and E) were analyzed for inflammasome effectors. Results are representative of three independent experiments. ( F and G ) Fadd −/− Ripk3 −/− BMDCs were transfected with control or Atg-5–specific siRNAs for 24 hours, and then Atg5 contents were determined (F). Cells were then treated with LPS + IETD for 8 hours, before determining IL-1β production (G, left panel) and LDH release (G, right panel). ( H ) Beclin-1, RIPK3, and FADD contents in BMDMs of indicated Becn1 genotypes were determined. ( I ) BMDMs were treated with LPS (0.1 μg/ml) + IETD (10 μM) for 18 hours, and then IL-1β levels were determined by ELISA (upper panel). LDH release was quantitated (lower panel). Data represent three [A, B, G (LDH), and I] or four [C and G (IL-1β)] independent experiments, and mean values are indicated. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001 for two-way ANOVA followed by a Sidak’s multiple comparison test.

Article Snippet: Anti-mouse RIPK3 (95702, RRID:AB_2721823), anti-pIKK (2078, RRID:AB_2079379), anti-IKKβ (8943, RRID:AB_11024092), anti-pJNK (9251, RRID:AB_331659), anti-JNK (9252, RRID:AB_2250373), anti-pp38 (9211, RRID:AB_331641), anti-p38 (9218, RRID:AB_10694846), anti-pERK (9101, RRID:AB_331646), anti-ERK (9102, RRID:AB_330744), anti-Atg5 (12994, RRID:AB_2630393), anti–Beclin-1 (3495, RRID:AB_1903911), anti–caspase-3 (9662, RRID:AB_331439), anti–cleaved caspase-3 (9661, RRID:AB_2341188), anti-mouse caspase-8 (4927, RRID:AB_2068301), and anti–cleaved caspase-8 (9429, RRID:AB_2068300) were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Transfection, Enzyme-linked Immunosorbent Assay

Journal: Science Advances

Article Title: Caspase-8 inactivation drives autophagy-dependent inflammasome activation in myeloid cells

doi: 10.1126/sciadv.abn9912

Figure Lengend Snippet: ( A and B ) A cathepsin-B inhibitor suppresses IETD-induced IL-1β generation and cell death in Fadd −/− Ripk3 −/− BMDCs. Fadd +/− Ripk3 −/− and Fadd −/− Ripk3 −/− BMDCs were stimulated with LPS (0.1 μg/ml) + IETD (15 μM) in the presence of CA-074Me (50 μM) or pepstatin A (50 μM, a cathepsin-D inhibitor) for 8 hours. IL-1β production was determined (A), and cell death (as assessed by LDH release) was quantified (B). * P < 0.05 and *** P < 0.001 for unpaired t test. ( C and D ) Cathepsin-B knockdown reduces IETD-induced IL-1β in Fadd −/− Ripk3 −/− BMDCs. Fadd −/− Ripk3 −/− BMDCs were transfected with control or cathepsin-B–specific siRNAs, before determining cathepsin-B levels (C). Cells were then treated with LPS (0.1 μg/ml) + IETD (10 μM) for 8 hours, before IL-1β production was quantitated (D). ** P < 0.01 for two-way ANOVA followed by Sidak’s multiple comparisons test. ( E ) Inhibition of cathepsin-B prevents IETD-triggered caspase-1/11 and GSDMD processing in Fadd −/− Ripk3 −/− BMDCs. BMDCs were stimulated with LPS (0.1 μg/ml) plus IETD (10 μM) for 6 hours. Culture supernatants (SUP) were precipitated, and whole-cell lysates (WCL) were collected and analyzed for caspase-1 p20, IL-1β p17, GSDMD-NT, procaspase-1, caspase-11, GSDMD, and pro–IL-1β. The experiment (E) was independently repeated three times with similar results.

Article Snippet: Anti-mouse RIPK3 (95702, RRID:AB_2721823), anti-pIKK (2078, RRID:AB_2079379), anti-IKKβ (8943, RRID:AB_11024092), anti-pJNK (9251, RRID:AB_331659), anti-JNK (9252, RRID:AB_2250373), anti-pp38 (9211, RRID:AB_331641), anti-p38 (9218, RRID:AB_10694846), anti-pERK (9101, RRID:AB_331646), anti-ERK (9102, RRID:AB_330744), anti-Atg5 (12994, RRID:AB_2630393), anti–Beclin-1 (3495, RRID:AB_1903911), anti–caspase-3 (9662, RRID:AB_331439), anti–cleaved caspase-3 (9661, RRID:AB_2341188), anti-mouse caspase-8 (4927, RRID:AB_2068301), and anti–cleaved caspase-8 (9429, RRID:AB_2068300) were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Transfection, Inhibition