caspase 11 inhibitor wedelolactone  (MedChemExpress)

Journal: JCI Insight

Article Title: CLEC5A is critical in Pseudomonas aeruginosa –induced NET formation and acute lung injury

doi: 10.1172/jci.insight.156613

Figure Lengend Snippet: ( A and B ) Neutrophils from WT, Clec5a –/– , Tlr2 –/– , and Tlr4 –/– mice were stimulated with LPS from E . coli, Klebsiella pneumoniae ( K.p ), and P . aeruginosa PAO1 (10 μg/mL) in the presence or absence of the antibiotic polymyxin B (10 μg/mL) ( n = 5 independent experiments) ( A ), or live and UV-killed P . aeruginosa PAO1 (MOI = 3 or 10) at 37°C for 1 hour ( n = 3 independent experiments) ( B ). ( C ) Mouse BM-derived neutrophils (5 × 10 5 /mL) were incubated with live PAO1 or UV-killed PAO1 (MOI = 3 or 10) for 1 hour at 37°C. For inflammasome activation, neutrophils were primed with LPS from E . coli (0.5 μg/mL) for 3 hours and then stimulated with ATP (3 mM) or nigericin (10 μM) for 45 minutes at 37°C. Western blots of cell lysates (20 μg) were probed with anti-GSDMD Ab, anti–caspase-1(CASP1) p20 Ab, anti–caspase-11 (CASP11) Ab, or anti-GAPDH Ab. Arrows indicate the location of caspase-1 p20. ( D and E ) Neutrophils from WT and Clec5a –/– mice were incubated with PAO1 (MOI = 10) for 1 hour at 37°C; caspase-1 (CASP1) cleavage was determined by western blotting ( D ), and images ( n = 3) were quantified using ImageJ (NIH) software. Western blot analyses were repeated for 3 times ( E ). ( F ) WT neutrophils were preincubated with DMSO, caspase-1 inhibitor Z-WEHD-FMK (20 μM), or caspase-11 inhibitor wedelolactone (100 μM) for 1 hour at room temperature, and they were further incubated with UV-killed or live PAO1 (MPO = 10) for 1 hour at 37°C ( n = 3 independent experiments). The level of NETs was calculated using the area (μm 2 ) of histone overlapping with MPO by MetaMorph software. ( G ) Cytokine/chemokine in culture media was determined by ELISA. Data are presented as mean ± SEM. Statistical test for A and B were measured with 2-way ANOVA, and E – G were calculated with an unpaired and nonparametric Student’s t test with Mann-Whitney U test. **** P < 0.0001.

Article Snippet: Caspase-1 inhibitor Z-WEHD-FMK (catalog FMK002) was purchased from R&D Systems, and caspase-11 inhibitor wedelolactone (catalog W4016) was purchased from MilliporeSigma.

Techniques: Derivative Assay, Incubation, Activation Assay, Western Blot, Software, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Journal: JCI Insight

Article Title: CLEC5A is critical in Pseudomonas aeruginosa –induced NET formation and acute lung injury

doi: 10.1172/jci.insight.156613

Figure Lengend Snippet: ( A and B ) BMDMs from WT ( n = 6), Clec5a –/– ( n = 6), Tlr2 –/– ( n = 4), and Tlr4 –/– ( n = 4) mice were stimulated with LPS from E.coli, Klebsiella pneumoniae ( K.p ), and PAO1 (100 ng/mL) ( A ), or they were incubated with live or UV-killed P . aeruginosa PAO1 strain (MOI = 3 or 10) ( B ) at 37°C for 1 hour. After washing, with serum-free RPMI containing 10 μg/mL gentamicin, cells were cultured in fresh RPMI containing 10% (v/v) FBS for 24 hours at 37°C. The levels of cytokine were measured by ELISA. Data were collected from 6 independent experiments. ( C ) BMDMs from WT mice were primed with Pam3CsK4 (1 μg/mL) for 4 hours at 37°C, followed by incubation with UV-killed or live P . aeruginosa PAO1 strain (MOI = 3 or 10) or transfected (T) with LPS from E . coli (2.5 μg/mL) for 15 hours at 37°C. Western blots were incubated with anti-GSDMD Ab, anti–caspase-11 (CASP11) Ab, anti–caspase-1 (CASP1) Ab, anti–caspase-3 (CASP3) Ab, or anti-GAPDH Ab. ( D and E ) BMDMs from WT and Clec5a –/– mice were incubated with live P . aeruginosa PAO1 strain (MOI = 10) for 15 hours at 37°C. Western blots of cell lysates were probed with anti-GSDMD Ab, anti–caspase-1 Ab, or anti-GAPDH Ab. Cleaved caspase-1 (cleaved CASP1) and cleaved GSDMD are shown as fold change compared with WT. LPS (T), transfection of E . coli LPS. Representative immunoblot of 4 independent experiments. Data are mean ± SEM, and statistical analysis for A and B were performed with 2-way ANOVA and by unpaired and nonparametric Student’s t test with Mann-Whitney U test for E . * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Caspase-1 inhibitor Z-WEHD-FMK (catalog FMK002) was purchased from R&D Systems, and caspase-11 inhibitor wedelolactone (catalog W4016) was purchased from MilliporeSigma.

Techniques: Incubation, Cell Culture, Enzyme-linked Immunosorbent Assay, Transfection, Western Blot, MANN-WHITNEY