Journal: Oncology Letters

Article Title: Distinct characteristics of dasatinib-induced pyroptosis in gasdermin E-expressing human lung cancer A549 cells and neuroblastoma SH-SY5Y cells

doi: 10.3892/ol.2020.11556

Figure Lengend Snippet: Changes in pyroptosis-related proteins detected with western blotting. SH-SY5Y cells were treated with (A) DOX or (B) dasatinib for 24 h, and (C) A549 cells were exposed to dasatinib for 48 h. One representative result from three independent experiments is shown. (D) Relative amounts of protein levels were quantified. *P<0.05, **P<0.01 represents the drug treated groups vs. control group. DOX, doxorubicin; Ctrl, control; GSDME, gasdermin E; GSDME-N N-terminal fragment of gasdemin E; GSDMD, gasdermin D; GSDMD-N, N-terminal fragment of gasdemin D; CASP3-C, cleaved caspase-3; PARP-1, poly (ADP-ribose) polymerase 1; PARP-1-C, cleaved fragment of PARP-1.

Article Snippet: Doxorubicin (DOX), specific caspase-3 inhibitor Z-DEVD-FMK (zDEVD) and pan-caspase inhibitor Z-VAD (OMe)-FMK (zVAD) were obtained from MedChemExpress.

Techniques: Western Blot, Control

Journal: Oncology Letters

Article Title: Distinct characteristics of dasatinib-induced pyroptosis in gasdermin E-expressing human lung cancer A549 cells and neuroblastoma SH-SY5Y cells

doi: 10.3892/ol.2020.11556

Figure Lengend Snippet: Requirement of caspase activation in dasatinib-induced pyroptosis. (A) Suppression of GSDME cleavage by pretreatment with caspase-3 inhibitor zDEVD when the SH-SY5Y cells were treated with 40 µm dasatinib. (B) Caspase-3 activity in A549 cells could not be inhibited by caspase-3 specific inhibitor zDEVD. (C) Inhibition of GSDME cleavage by pan-caspase inhibitor zVAD when the A549 cells were treated with 30 µm dasatinib. One representative result from three independent experiments is shown. *P<0.05, **P<0.01 represents the drug treated groups vs. control group. GSDME, gasdermin E; GSDME-N, N-terminal fragment of GSDME; zDEVD, caspase-3 inhibitor Z-DEVD-FMK; zVAD, pan-caspase inhibitor Z-VAD (OMe)-FMK; CASP3-C, cleaved caspase-3.

Article Snippet: Doxorubicin (DOX), specific caspase-3 inhibitor Z-DEVD-FMK (zDEVD) and pan-caspase inhibitor Z-VAD (OMe)-FMK (zVAD) were obtained from MedChemExpress.

Techniques: Activation Assay, Activity Assay, Inhibition, Control

Journal: The Journal of Experimental Medicine

Article Title: EEF2-inactivating toxins engage the NLRP1 inflammasome and promote epithelial barrier disruption

doi: 10.1084/jem.20230104

Figure Lengend Snippet: P. aeruginosa triggers human NLRP1 inflammasome activation in corneal and nasal epithelial cells. (A) Cell lysis (LDH) and IL-1β/IL-18 release evaluation in pHCECs and pHNECs upon P. aeruginosa (PAO1, 1.10 5 bacteria) co-culture for 24 h. When specified, the pan Caspase inhibitor (Z-VAD, 20 µM), Caspase-1 inhibitor (Z-YVAD, 20 µM), Caspase-3/7 inhibitor (Z-DEVD, 20 µM), and Caspase-8 inhibitor (Z-IETD, 20 µM) were used. ***P ≤ 0.001, two-way ANOVA with multiple comparisons. Values are expressed as mean ± SEM. Graphs show one experiment performed in triplicates at least three times. (B) Immunoblotting examination of NLRP1, NLRP3, and Tubulin in resting, PAO1-exposed as in A or LPS-primed pHCECs and pHNECs or in pHCECs and pHNECs genetically invalidated for NLRP1 using CRISPR-Cas9. PMA (100 µg/ml)- or LPS (100 ng/ml)-primed THP1 monocytic cell line was used as a positive control for NLRP3 expression. Immunoblots show lysates from one experiment performed three times. (C) Florescence microscopy and associated quantifications of ASC-GFP specks in A549 NLRP1+/ASC-GFP and A549 NLRP1−/ASC-GFP reporter cell lines exposed to P. aeruginosa (PAO1, 1.10 5 bacteria) for 24 h. ASC-GFP (green) pictures were taken in the dish after the infection. Images shown are from one experiment and are representative of n = 3 independent experiments; scale bars, 10 µm. ASC complex percentage was performed by determining the ratios of cells positive for ASC speckles on the total nuclei (Hoechst). At least 10 fields from each experiment were analyzed. Values are expressed as mean ± SEM. ***P ≤ 0.001, one-way ANOVA. (D) Immunoblotting characterization of genetic invalidation of NLRP1 in pHCECs and pHNECs population using CRISPR-Cas9 and microscopy visualization of plasma membrane permeabilization (PI incorporation, orange) in pHCECs co-cultured with PAO1 (1.10 5 bacteria) for 24 h. (E) sgRNA CD8 (SgCD8) was used as control and served as WT cells during subsequent experiments described in E. Images shown are from one experiment and are representative of n = 3 independent experiments; scale bars, 20 µm. Cell lysis (LDH), IL-18 release, and CFU evaluation in WT (SgCD8, D) or NLRP1 -deficient pHCECs and pHNECs, upon VbP (15 µM) treatment or P. aeruginosa (PAO1, 1.10 5 bacteria) co-culture for 24 h. For CFU analysis 1 × 10 4 (MOI 1) or 1 × 10 5 (MOI 10) bacteria were used. ***P ≤ 0.001, two-way ANOVA with multiple comparisons. Values are expressed as mean ± SEM. Graphs show one experiment performed in triplicates at least three times. Source data are available for this figure: .

Article Snippet: Caspase-3/7 inhibitor ZDEVD-FMK 20 µM , S7312 , Selleck.

Techniques: Activation Assay, Lysis, Bacteria, Co-Culture Assay, Western Blot, CRISPR, Positive Control, Expressing, Microscopy, Infection, Membrane, Cell Culture

Journal: The Journal of Experimental Medicine

Article Title: EEF2-inactivating toxins engage the NLRP1 inflammasome and promote epithelial barrier disruption

doi: 10.1084/jem.20230104

Figure Lengend Snippet: List of reagents used in the study

Article Snippet: Caspase-3/7 inhibitor ZDEVD-FMK 20 µM , S7312 , Selleck.

Techniques: Protease Inhibitor, Staining, Marker, CyQUANT Assay, Mutagenesis, Membrane, Recombinant

Journal: Heliyon

Article Title: Pyroptotic cell death by exposure to 1-butanol in H9c2 cardiomyoblastoma cells

doi: 10.1016/j.heliyon.2020.e05503

Figure Lengend Snippet: Casapse3 activation and cleavage of GSDMD and GSDME during exposure of H9c2 cells to 1-butanol. ( A and B) Time-dependent activation of caspase-3 and proteolysis of GSDMD. H9c2 cells were exposed to 150 mM 1-butanol for 1–6 h, and immunoblot analyses of caspase-1, cleaved caspase-3, and GSDMD were performed. ( C and D) Concentration-dependency of the activation of caspase-3 and proteolysis of GSDMD. H9c2 cells were exposed to 100–200 mM 1-butanol for 6 h, and immunoblot analyses of caspase-1, cleaved caspase-3, and GSDMD were performed. (E) GSDME cleavage into p30 fragment in 1-butanol-treated cells. Con, control. H9c2 cells were exposed to 150 mM 1-butanol for 6 h, and immunoblot analyses of GSDME was performed. FL indicates the full-length forms of caspase-1, GSDMD, or GSDME. #, uncharacterized fragment. The relative intensities of the bands were quantified by densitometry and normalized to actin. The graph shows mean ± S.D. (n = 4). ∗∗p < 0.01 versus control (Con). ( F ) Cells were exposed to 150 mM 1-butanol for 6 h in the absence or presence of 10 μM zDEVD and LDH release were assessed. The graph shows mean ± S.D. (n = 3). ∗∗p < 0.01.

Article Snippet: Rho-kinase inhibitor Y-27632 (10 μM, Wako Pure Chemicals) and caspase-3 inhibitor z-DEVD-FMK (zDEVD, 10 μM, MBL, Nagoya, Japan) was added to the medium 30 min before treatment with 1-butanol.

Techniques: Activation Assay, Western Blot, Concentration Assay

Journal: Heliyon

Article Title: Pyroptotic cell death by exposure to 1-butanol in H9c2 cardiomyoblastoma cells

doi: 10.1016/j.heliyon.2020.e05503

Figure Lengend Snippet: Casapse3 activation and cleavage of GSDME during exposure of J774.1 cells to 1-butanol. ( A) Concentration-dependent changes in plasma membrane morphology during exposure of J774.1 cells to 1-butanol. Cells were treated with 1, 10, 100 or 200 mM 1-butanol for 6 h and morphological changes were examined under a light microscope. White arrows indicate ballooning. ( B) Time-dependent activation of caspase-3 and proteolysis of GSDME. J774.1 cells were exposed to 1, 10, 100 or 200 mM 1-butanol for 1, 3 and 6 h, and immunoblot analyses of cleaved caspase-3 and GSDME were performed. C, control. FL indicates the full-length forms of GSDME.

Article Snippet: Rho-kinase inhibitor Y-27632 (10 μM, Wako Pure Chemicals) and caspase-3 inhibitor z-DEVD-FMK (zDEVD, 10 μM, MBL, Nagoya, Japan) was added to the medium 30 min before treatment with 1-butanol.

Techniques: Activation Assay, Concentration Assay, Light Microscopy, Western Blot

Journal: BMC Cancer

Article Title: 5-allyl-7-gen-difluoromethoxychrysin enhances TRAIL-induced apoptosis in human lung carcinoma A549 cells

doi: 10.1186/1471-2407-11-322

Figure Lengend Snippet: Effects of AFMC and/or TRAIL on the caspase activities of A549 cells . Figure 2A: Enzymatic activities of caspase -3, -8 and -9 were determined by incubation of 20 μg of total protein with 200 μmol/L chromogenic substrate (Ac-DEVD-pNA, Ac-IETD-pNA or Ac-LEHD-pNA) in 100 μL of assay buffer for 2 h at 37°C. The release of the chromophore p-nitroanilide (pNA) was monitored spectrophotometrically at 405 nm. Data are shown as mean ± S.D. (n = 3). a P < 0.05 vs. 0.2% DMSO; b P < 0.05 vs. 30 ng/mL TRAIL or 1.0 μmol/L AFMC alone. Figure 2B: A549 cells were pretreated with 1.0 μmol/L AFMC for 30 min and then treated with 30 ng/mL TRAIL for 24 h in the presence or absence of 10.0 μmol/L caspase inhibitors. Enzymatic activities of caspase -3, -8 and -9 were determined as described above. Data are shown as mean ± S.D. (n = 3). a P < 0.05 vs. 0.2% DMSO; b P < 0.05 vs. 30 ng/mL TRAIL or 1.0 μmol/L AFMC alone. Figure 2C: A549 cells were treated as described above. The frequency of cells in the sub-G1 phase was measured using flow cytometry. Data are shown as mean ± S.D. (n = 3). a P < 0.05 vs. 0.2% DMSO; b P < 0.05 vs. pretreatment with caspase inhibitors.

Article Snippet: DR5/Fc chimera protein and caspase inhibitors (zDEVD-fmk for caspase-3, zIETD-fmk for caspase-8 and zLEHD-fmk for caspase-9) were from R&D Systems (Minneapolis, MN).

Techniques: Incubation, Flow Cytometry

Journal: BMC Cancer

Article Title: 5-allyl-7-gen-difluoromethoxychrysin enhances TRAIL-induced apoptosis in human lung carcinoma A549 cells

doi: 10.1186/1471-2407-11-322

Figure Lengend Snippet: Effects of DR5/Fc and DR5 siRNA on AFMC-induced DR5 expression and AFMC/TRAIL-induced apoptosis . Figure 4A: A549 cells were transfected with LacZ siRNA or siRNA against DR5 followed by treatment with 1.0 μmol/L AFMC for 24 h. DR5 levels were measured by Western blotting. β-actin was used as a loading control. Figure 4B: A549 cells were preincubated with 1.0 μg/mL DR5-specific blocking chimera antibody or transfected with siRNAs, incubated for 24 h, and then treated with or without 1.0 μmol/L AFMC for 30 min followed by treatment with 30 ng/mL TRAIL for 24 h. The frequency of cells in the sub-G1 phase was measured using flow cytometry. Data are shown as mean ± S.D. (n = 3). aP < 0.05 vs. 0.2% DMSO; bP < 0.05 vs. combined treatment. Figure 4C: A549 cells were treated as described above. Enzymatic activity of caspase-3 was determined by incubation of 20 μg of total protein with 200 μM of Ac-DEVD-pNA in 100 μL of assay buffer for 2 h at 37°C. pNA release was monitored spectrophotometrically at 405 nm. Data are shown as mean ± S.D. (n = 3). aP < 0.05 vs. 0.2% DMSO; bP < 0.05 vs. combined treatment. Figure 4D: A549 cells were treated as described above. Enzymatic activity of caspase-8 was determined by incubation of 20 μg of total protein with 200 μM of Ac-IETD-pNA in 100 μL of assay buffer for 2 h at 37°C. pNA release was monitored spectrophotometrically at 405 nm. Data are shown as mean ± S.D. (n = 3). aP < 0.05 vs. 0.2% DMSO; bP < 0.05 vs. combined treatment.

Article Snippet: DR5/Fc chimera protein and caspase inhibitors (zDEVD-fmk for caspase-3, zIETD-fmk for caspase-8 and zLEHD-fmk for caspase-9) were from R&D Systems (Minneapolis, MN).

Techniques: Expressing, Transfection, Western Blot, Control, Blocking Assay, Incubation, Flow Cytometry, Activity Assay

Journal: BMC Cancer

Article Title: 5-allyl-7-gen-difluoromethoxychrysin enhances TRAIL-induced apoptosis in human lung carcinoma A549 cells

doi: 10.1186/1471-2407-11-322

Figure Lengend Snippet: AFMC neither induce expression of DR5 nor enhance TRAIL-induced apoptosis in WI-38 cells . Figure 6A: WI-38 cells were treated with AFMC for 30 min and then treated with TRAIL for 24 h at the indicated concentrations. Cytotoxicity was assessed by ELISA. Columns, average of three individual experiments; bars, SD. a P < 0.05 vs. 0.2% DMSO; c P < 0.05 vs. 1.0 μmol/L AFMC. Figure 6B: WI-38 cells were treated with 1.0 μmol/L AFMC, 30 ng/mL TRAIL or the pretreated with 1.0 μmol/L AFMC for 30 min followed by 30 ng/mL TRAIL for 24 h. DNA content of the cells was analyzed by flow cytometry. Figure 6C: WI-38 cells were treated as described above. The enzymatic activity of caspase-3 was determined by incubation of 20 μg total protein with 200 μM of Ac-DEVD-pNA in 100 μL of assay buffer for 2 h at 37°C. pNA release was monitored spectrophotometrically at 405 nm. Figure 6D: WI-38 cells were treated as described above. The enzymatic activity of caspase-8 was determined by incubation of 20 μg of total protein with 200 μM of Ac-IETD-pNA in 100 μL of assay buffer for 2 h at 37°C. pNA release was monitored spectrophotometrically at 405 nm. Data are shown as mean ± S.D. (n = 3). Figure 6E: WI-38 cells were treated as described above. Fragmented DNA was extracted from the treated cells and analyzed on a 2.0% agarose gel. Figure 6F: WI-38 cells were treated with the indicated concentrations of AFMC for 24 h, and DR5 levels were assessed by Western blotting. β-actin was used as a loading control.

Article Snippet: DR5/Fc chimera protein and caspase inhibitors (zDEVD-fmk for caspase-3, zIETD-fmk for caspase-8 and zLEHD-fmk for caspase-9) were from R&D Systems (Minneapolis, MN).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Activity Assay, Incubation, Agarose Gel Electrophoresis, Western Blot, Control

Journal:

Article Title: The CB1/VR1 agonist arvanil induces apoptosis through an FADD/caspase-8-dependent pathway

doi: 10.1038/sj.bjp.0705532

Figure Lengend Snippet: Schematic diagram highlighting the proposed mechanism for arvanil-induced apoptosis. Arvanil induces FADD clustering, which allows the recruitment of caspase-8. Then, this caspase is cleaved and activated, leading to an apoptotic cascade that includes Bid truncation, leaking of cytochrome c from mitochondria and activation of different effector caspases (-3, -7, -9). To complement this pathway, arvanil induces ROS that may help to DISC formation and may also induce the mitochondrial release of cytochrome c.

Article Snippet: The cell-permeable inhibitors of caspases zDEVD-fmk and zIETD-fmk ( N -benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethyl ketone and N -benzyloxycarbonyl-Ile-Glu-Thr-Asp-fluoromethyl ketone, respectively) were from Bachem (Bubendorf, Switzerland).

Techniques: Activation Assay