via fluor cell proliferation kit Search Results


tacs tdt in situ apoptosis detection kit  (R&D Systems)
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R&D Systems tacs tdt in situ apoptosis detection kit
Tacs Tdt In Situ Apoptosis Detection Kit, supplied by R&D Systems, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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antibodies for gsdmd  (Cell Signaling Technology Inc)
93
Cell Signaling Technology Inc antibodies for gsdmd
Fig. 2. CuB induced <t>NLRP3/caspase-1/GSDMD-mediated</t> pyroptosis. a The morphology changes of A549 cells after treatment with CuB (100 nM) for 8 h (Scale bar=25 µm). b The features of pyroptosis in A549 cells were detected by TEM (Scale bar=2 µm). c The expression of GSDMD-FL, GSDMD-N, pro-IL-1β, mature IL-1β, <t>and</t> <t>HMGB1</t> in A549 cells. d Immunofluorescence detection of the expression of HMGB1 in A549 cells (Scale bar=20 µm). e The EGFP-NLRP3 and OFPSpark-NEK7 fluorescence were examined by immunofluorescence assay (Scale bar=7.5 µm). f Co-IP analysis of the interaction between NEK7 and NLRP3 in A549 cells after treatment with CuB (100 nM) for 24 h in the presence of VX765 (60 µm). g The expression of NLRP3, NEK7, ASC, pro-caspase-1, and cleaved caspase-1 in A549 cells. h, i Immunofluorescence detection of the caspase-1 expression (Scale bar=25 µm) and ASC dot formation in A549 cells (Scale bar=7.5 µm). j Annexin V and 7-AAD double staining assay was used to confirm CuB (100 nM)-induced pyroptosis in A549 cells.
Antibodies For Gsdmd, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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click-it plus edu alexa fluor 647 flow cytometry assay kit  (Thermo Fisher)
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Thermo Fisher click-it plus edu alexa fluor 647 flow cytometry assay kit
Fig. 2. CuB induced <t>NLRP3/caspase-1/GSDMD-mediated</t> pyroptosis. a The morphology changes of A549 cells after treatment with CuB (100 nM) for 8 h (Scale bar=25 µm). b The features of pyroptosis in A549 cells were detected by TEM (Scale bar=2 µm). c The expression of GSDMD-FL, GSDMD-N, pro-IL-1β, mature IL-1β, <t>and</t> <t>HMGB1</t> in A549 cells. d Immunofluorescence detection of the expression of HMGB1 in A549 cells (Scale bar=20 µm). e The EGFP-NLRP3 and OFPSpark-NEK7 fluorescence were examined by immunofluorescence assay (Scale bar=7.5 µm). f Co-IP analysis of the interaction between NEK7 and NLRP3 in A549 cells after treatment with CuB (100 nM) for 24 h in the presence of VX765 (60 µm). g The expression of NLRP3, NEK7, ASC, pro-caspase-1, and cleaved caspase-1 in A549 cells. h, i Immunofluorescence detection of the caspase-1 expression (Scale bar=25 µm) and ASC dot formation in A549 cells (Scale bar=7.5 µm). j Annexin V and 7-AAD double staining assay was used to confirm CuB (100 nM)-induced pyroptosis in A549 cells.
Click It Plus Edu Alexa Fluor 647 Flow Cytometry Assay Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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anti-panmhc-i antibody w6/32  (Bio X Cell)
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Bio X Cell anti-panmhc-i antibody w6/32
Fig. 2. CuB induced <t>NLRP3/caspase-1/GSDMD-mediated</t> pyroptosis. a The morphology changes of A549 cells after treatment with CuB (100 nM) for 8 h (Scale bar=25 µm). b The features of pyroptosis in A549 cells were detected by TEM (Scale bar=2 µm). c The expression of GSDMD-FL, GSDMD-N, pro-IL-1β, mature IL-1β, <t>and</t> <t>HMGB1</t> in A549 cells. d Immunofluorescence detection of the expression of HMGB1 in A549 cells (Scale bar=20 µm). e The EGFP-NLRP3 and OFPSpark-NEK7 fluorescence were examined by immunofluorescence assay (Scale bar=7.5 µm). f Co-IP analysis of the interaction between NEK7 and NLRP3 in A549 cells after treatment with CuB (100 nM) for 24 h in the presence of VX765 (60 µm). g The expression of NLRP3, NEK7, ASC, pro-caspase-1, and cleaved caspase-1 in A549 cells. h, i Immunofluorescence detection of the caspase-1 expression (Scale bar=25 µm) and ASC dot formation in A549 cells (Scale bar=7.5 µm). j Annexin V and 7-AAD double staining assay was used to confirm CuB (100 nM)-induced pyroptosis in A549 cells.
Anti Panmhc I Antibody W6/32, supplied by Bio X Cell, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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cell counting kit-8 cck-8  (Abbkine Inc)
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Abbkine Inc cell counting kit-8 cck-8
Fig. 2. CuB induced <t>NLRP3/caspase-1/GSDMD-mediated</t> pyroptosis. a The morphology changes of A549 cells after treatment with CuB (100 nM) for 8 h (Scale bar=25 µm). b The features of pyroptosis in A549 cells were detected by TEM (Scale bar=2 µm). c The expression of GSDMD-FL, GSDMD-N, pro-IL-1β, mature IL-1β, <t>and</t> <t>HMGB1</t> in A549 cells. d Immunofluorescence detection of the expression of HMGB1 in A549 cells (Scale bar=20 µm). e The EGFP-NLRP3 and OFPSpark-NEK7 fluorescence were examined by immunofluorescence assay (Scale bar=7.5 µm). f Co-IP analysis of the interaction between NEK7 and NLRP3 in A549 cells after treatment with CuB (100 nM) for 24 h in the presence of VX765 (60 µm). g The expression of NLRP3, NEK7, ASC, pro-caspase-1, and cleaved caspase-1 in A549 cells. h, i Immunofluorescence detection of the caspase-1 expression (Scale bar=25 µm) and ASC dot formation in A549 cells (Scale bar=7.5 µm). j Annexin V and 7-AAD double staining assay was used to confirm CuB (100 nM)-induced pyroptosis in A549 cells.
Cell Counting Kit 8 Cck 8, supplied by Abbkine Inc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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alexa fluor™ 488 dye  (Thermo Fisher)
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Thermo Fisher alexa fluor™ 488 dye
Fig. 2. CuB induced <t>NLRP3/caspase-1/GSDMD-mediated</t> pyroptosis. a The morphology changes of A549 cells after treatment with CuB (100 nM) for 8 h (Scale bar=25 µm). b The features of pyroptosis in A549 cells were detected by TEM (Scale bar=2 µm). c The expression of GSDMD-FL, GSDMD-N, pro-IL-1β, mature IL-1β, <t>and</t> <t>HMGB1</t> in A549 cells. d Immunofluorescence detection of the expression of HMGB1 in A549 cells (Scale bar=20 µm). e The EGFP-NLRP3 and OFPSpark-NEK7 fluorescence were examined by immunofluorescence assay (Scale bar=7.5 µm). f Co-IP analysis of the interaction between NEK7 and NLRP3 in A549 cells after treatment with CuB (100 nM) for 24 h in the presence of VX765 (60 µm). g The expression of NLRP3, NEK7, ASC, pro-caspase-1, and cleaved caspase-1 in A549 cells. h, i Immunofluorescence detection of the caspase-1 expression (Scale bar=25 µm) and ASC dot formation in A549 cells (Scale bar=7.5 µm). j Annexin V and 7-AAD double staining assay was used to confirm CuB (100 nM)-induced pyroptosis in A549 cells.
Alexa Fluor™ 488 Dye, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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cleaved caspase 3  (Cell Signaling Technology Inc)
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Cell Signaling Technology Inc cleaved caspase 3
Fig. 2. CuB induced <t>NLRP3/caspase-1/GSDMD-mediated</t> pyroptosis. a The morphology changes of A549 cells after treatment with CuB (100 nM) for 8 h (Scale bar=25 µm). b The features of pyroptosis in A549 cells were detected by TEM (Scale bar=2 µm). c The expression of GSDMD-FL, GSDMD-N, pro-IL-1β, mature IL-1β, <t>and</t> <t>HMGB1</t> in A549 cells. d Immunofluorescence detection of the expression of HMGB1 in A549 cells (Scale bar=20 µm). e The EGFP-NLRP3 and OFPSpark-NEK7 fluorescence were examined by immunofluorescence assay (Scale bar=7.5 µm). f Co-IP analysis of the interaction between NEK7 and NLRP3 in A549 cells after treatment with CuB (100 nM) for 24 h in the presence of VX765 (60 µm). g The expression of NLRP3, NEK7, ASC, pro-caspase-1, and cleaved caspase-1 in A549 cells. h, i Immunofluorescence detection of the caspase-1 expression (Scale bar=25 µm) and ASC dot formation in A549 cells (Scale bar=7.5 µm). j Annexin V and 7-AAD double staining assay was used to confirm CuB (100 nM)-induced pyroptosis in A549 cells.
Cleaved Caspase 3, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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beyoclickedu cell proliferation kit with alexa fluor assay  (Beyotime)
90
Beyotime beyoclickedu cell proliferation kit with alexa fluor assay
Fig. 2. CuB induced <t>NLRP3/caspase-1/GSDMD-mediated</t> pyroptosis. a The morphology changes of A549 cells after treatment with CuB (100 nM) for 8 h (Scale bar=25 µm). b The features of pyroptosis in A549 cells were detected by TEM (Scale bar=2 µm). c The expression of GSDMD-FL, GSDMD-N, pro-IL-1β, mature IL-1β, <t>and</t> <t>HMGB1</t> in A549 cells. d Immunofluorescence detection of the expression of HMGB1 in A549 cells (Scale bar=20 µm). e The EGFP-NLRP3 and OFPSpark-NEK7 fluorescence were examined by immunofluorescence assay (Scale bar=7.5 µm). f Co-IP analysis of the interaction between NEK7 and NLRP3 in A549 cells after treatment with CuB (100 nM) for 24 h in the presence of VX765 (60 µm). g The expression of NLRP3, NEK7, ASC, pro-caspase-1, and cleaved caspase-1 in A549 cells. h, i Immunofluorescence detection of the caspase-1 expression (Scale bar=25 µm) and ASC dot formation in A549 cells (Scale bar=7.5 µm). j Annexin V and 7-AAD double staining assay was used to confirm CuB (100 nM)-induced pyroptosis in A549 cells.
Beyoclickedu Cell Proliferation Kit With Alexa Fluor Assay, supplied by Beyotime, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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vcam-1 duoset kit  (R&D Systems)
90
R&D Systems vcam-1 duoset kit
Fig. 2. CuB induced <t>NLRP3/caspase-1/GSDMD-mediated</t> pyroptosis. a The morphology changes of A549 cells after treatment with CuB (100 nM) for 8 h (Scale bar=25 µm). b The features of pyroptosis in A549 cells were detected by TEM (Scale bar=2 µm). c The expression of GSDMD-FL, GSDMD-N, pro-IL-1β, mature IL-1β, <t>and</t> <t>HMGB1</t> in A549 cells. d Immunofluorescence detection of the expression of HMGB1 in A549 cells (Scale bar=20 µm). e The EGFP-NLRP3 and OFPSpark-NEK7 fluorescence were examined by immunofluorescence assay (Scale bar=7.5 µm). f Co-IP analysis of the interaction between NEK7 and NLRP3 in A549 cells after treatment with CuB (100 nM) for 24 h in the presence of VX765 (60 µm). g The expression of NLRP3, NEK7, ASC, pro-caspase-1, and cleaved caspase-1 in A549 cells. h, i Immunofluorescence detection of the caspase-1 expression (Scale bar=25 µm) and ASC dot formation in A549 cells (Scale bar=7.5 µm). j Annexin V and 7-AAD double staining assay was used to confirm CuB (100 nM)-induced pyroptosis in A549 cells.
Vcam 1 Duoset Kit, supplied by R&D Systems, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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proteome profilerw human apoptosis array kit  (R&D Systems)
90
R&D Systems proteome profilerw human apoptosis array kit
Fig. 2. CuB induced <t>NLRP3/caspase-1/GSDMD-mediated</t> pyroptosis. a The morphology changes of A549 cells after treatment with CuB (100 nM) for 8 h (Scale bar=25 µm). b The features of pyroptosis in A549 cells were detected by TEM (Scale bar=2 µm). c The expression of GSDMD-FL, GSDMD-N, pro-IL-1β, mature IL-1β, <t>and</t> <t>HMGB1</t> in A549 cells. d Immunofluorescence detection of the expression of HMGB1 in A549 cells (Scale bar=20 µm). e The EGFP-NLRP3 and OFPSpark-NEK7 fluorescence were examined by immunofluorescence assay (Scale bar=7.5 µm). f Co-IP analysis of the interaction between NEK7 and NLRP3 in A549 cells after treatment with CuB (100 nM) for 24 h in the presence of VX765 (60 µm). g The expression of NLRP3, NEK7, ASC, pro-caspase-1, and cleaved caspase-1 in A549 cells. h, i Immunofluorescence detection of the caspase-1 expression (Scale bar=25 µm) and ASC dot formation in A549 cells (Scale bar=7.5 µm). j Annexin V and 7-AAD double staining assay was used to confirm CuB (100 nM)-induced pyroptosis in A549 cells.
Proteome Profilerw Human Apoptosis Array Kit, supplied by R&D Systems, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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cleaved notch1  (Cell Signaling Technology Inc)
90
Cell Signaling Technology Inc cleaved notch1
Reduced cilium length is caused by enhanced γ-secretase activity and diminished Foxj1a levels. (a) 2.5-fold increased γ-secretase activity in Rer1p-depleted CL4 cells as indicated by increased NICD production ( n = 7). ctrl, control. (b) Overexpression of NICD but not of Cadherin ICD (CICD) reduces cilium length in CL4 cells. (c) Nanomolar concentrations of γ-secretase inhibition (InhX) rescue cilia length in Rer1p-depleted CL4 cells without affecting cilia length in control cells. Acetylated tubulin staining ( n = 150; three experiments). NSC, nonspecific control. (d) Reduced deltaA but unaffected <t>notch1</t> and notch3 levels in 24-hpf SMO. (e) Enhanced Notch signaling in the ear sensory patch, but not in the tail, indicated by increased fluorescence of the Notch reporter line Tp1:hmgb1-mCherry. Quantification is shown of highlighted regions ( n > 15). UI, uninjected. (f) FM1-43 dye labeling shows a twofold reduction in the number of neuromasts in 3-dpf SMO. (g) Twofold reduction in the number of hair cells per neuromast (PLL) in 2-dpf SMO-injected ET4 transgenic embryos. Increased Notch signaling (100 pg NICD mRNA) results in less hair cells per neuromast (71% of control), whereas inhibiting Notch signaling (25 µM compound E [cpdE]) increases hair cell number (150% of control) and rescues the effect of SMO from 46 to 93.6% ( n > 12). (h) WISH shows normal differentiation of the DFC/KV lineage ( lrdr1 ) but reduced foxj1a levels in DFC (80% epiboly, arrows), KV (bud, arrows), pronephros (arrowheads), and notochord (asterisks). ss, somite stage. (i) Quantification of foxj1a levels by quantitative PCR in SMO. NICD does not affect foxj1a levels in the bud. NI, not injected. (j–l) Partial rescue of pronephros (j and k) and KV (l) cilia length upon coinjection of Foxj1a ( n ≥ 11 embryos; three experiments). Ac.tub, acetylated tubulin. Means ± SEM; *, P < 0.05; **, P < 0.01; ***, P < 0.001, Wilcoxon rank sum test (b and c) or Student’s t test (a, e, g, i, k, and l).
Cleaved Notch1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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click-it edu alexa fluor 647 flow cytometry assay kit  (Thermo Fisher)
90
Thermo Fisher click-it edu alexa fluor 647 flow cytometry assay kit
(A) Scheme of these experiments. (B, C) Example (B; scale bar, 100 µm) and quantification (C) of apoptotic cells found in the epidermis of mice of the indicated genotypes 24 h upon the application of DMBA ( n = 4). In (B), sections were stained with antibodies to the cleaved fragment of caspase 3 (ID number: 12367) and 14, 4′,6-diamidino-2-phenylindole (DAPI) to reveal apoptotic cells (red color) and cell nuclei (blue color), respectively. (D, E) Example (D; scale bar, 100 µm) and quantification (E) of phospho-histone H2AX + keratinocytes (brown color) present in the epidermis of short-term DMBA-treated mice of indicated genotypes ( n = 4). p-, phosphorylated. (F) Number of apoptotic (annexin V + ) wild-type and Vav2 −/− ; Vav3 −/− keratinocytes induced after 8 (left panel) and 12 (right panel) h in the indicated culture conditions ( n = 4). Bleo, bleomycin; DTT, dithiothreitol; NS, no statistically significant. (G, H) Example of a flow <t>cytometry</t> experiment (G) and subsequent quantification (H) of the level of apoptosis induced by either DMBA (G, H) or serum starvation (H) in wild-type and Vav2 −/− ; Vav3 −/− keratinocytes ectopically expressing GFP either alone (G, H) or in combination with HA-Vav2 (H) or Myc-Vav3 (G, H) ( n = 3). In (G), only the gated GFP + cells are shown.
Click It Edu Alexa Fluor 647 Flow Cytometry Assay Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Image Search Results


Fig. 2. CuB induced NLRP3/caspase-1/GSDMD-mediated pyroptosis. a The morphology changes of A549 cells after treatment with CuB (100 nM) for 8 h (Scale bar=25 µm). b The features of pyroptosis in A549 cells were detected by TEM (Scale bar=2 µm). c The expression of GSDMD-FL, GSDMD-N, pro-IL-1β, mature IL-1β, and HMGB1 in A549 cells. d Immunofluorescence detection of the expression of HMGB1 in A549 cells (Scale bar=20 µm). e The EGFP-NLRP3 and OFPSpark-NEK7 fluorescence were examined by immunofluorescence assay (Scale bar=7.5 µm). f Co-IP analysis of the interaction between NEK7 and NLRP3 in A549 cells after treatment with CuB (100 nM) for 24 h in the presence of VX765 (60 µm). g The expression of NLRP3, NEK7, ASC, pro-caspase-1, and cleaved caspase-1 in A549 cells. h, i Immunofluorescence detection of the caspase-1 expression (Scale bar=25 µm) and ASC dot formation in A549 cells (Scale bar=7.5 µm). j Annexin V and 7-AAD double staining assay was used to confirm CuB (100 nM)-induced pyroptosis in A549 cells.

Journal: Pharmacological research

Article Title: Cucurbitacin B inhibits non-small cell lung cancer in vivo and in vitro by triggering TLR4/NLRP3/GSDMD-dependent pyroptosis.

doi: 10.1016/j.phrs.2021.105748

Figure Lengend Snippet: Fig. 2. CuB induced NLRP3/caspase-1/GSDMD-mediated pyroptosis. a The morphology changes of A549 cells after treatment with CuB (100 nM) for 8 h (Scale bar=25 µm). b The features of pyroptosis in A549 cells were detected by TEM (Scale bar=2 µm). c The expression of GSDMD-FL, GSDMD-N, pro-IL-1β, mature IL-1β, and HMGB1 in A549 cells. d Immunofluorescence detection of the expression of HMGB1 in A549 cells (Scale bar=20 µm). e The EGFP-NLRP3 and OFPSpark-NEK7 fluorescence were examined by immunofluorescence assay (Scale bar=7.5 µm). f Co-IP analysis of the interaction between NEK7 and NLRP3 in A549 cells after treatment with CuB (100 nM) for 24 h in the presence of VX765 (60 µm). g The expression of NLRP3, NEK7, ASC, pro-caspase-1, and cleaved caspase-1 in A549 cells. h, i Immunofluorescence detection of the caspase-1 expression (Scale bar=25 µm) and ASC dot formation in A549 cells (Scale bar=7.5 µm). j Annexin V and 7-AAD double staining assay was used to confirm CuB (100 nM)-induced pyroptosis in A549 cells.

Article Snippet: Antibodies for GSDMD (#93709), GAPDH (#8884), HMGB1 (#3935), cleaved-IL-1β (#83186), NLRP3 (#15101), cleaved-caspase1 (#4199), ASC (#13833), IL-1β (#12703), Gasdermin D (#93709), Tom20 (#42406), and the secondary antibodies including horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (#7074) were purchased from Cell Signaling Technology (CST, Danvers, MA, USA).

Techniques: Expressing, Immunofluorescence, Fluorescence, Co-Immunoprecipitation Assay, Double Staining

Fig. 3. ROS promoted accumulation of pyroptosis-related Tom20. a JC-1 kit was used to examine the MMP levels in A549 cells (Scale bar=25 µm). b, c ROS level was analyzed by flow cytometry in A549 cells (n ≥3, ***P＜0.001 vs CuB; ###P＜0.001 vs control group). d Effect of NAC on morphological feature changes in A549 cells (Scale bar=25 µm). e Western blotting analysis of the expression of GSDMD-FL, GSDMD-N, pro-IL-1β, mature IL-1β, pro-caspase-1 and cleaved caspase-1 in A549 cells. f Annexin V and 7-AAD double staining assay was used to identify the inhibition of NAC CuB-induced pyroptosis by NAC in A549 cells. g Immunofluorescence detection of the expression of mitochondrial protein Tom20 in A549 cells (Scale bar=10 µm). h The cell viability in Tom 20 knockdown A549 cells (n ≥3, ***P＜0.001 vs CuB; ###P＜0.001 vs si-Ctrl group).

Journal: Pharmacological research

Article Title: Cucurbitacin B inhibits non-small cell lung cancer in vivo and in vitro by triggering TLR4/NLRP3/GSDMD-dependent pyroptosis.

doi: 10.1016/j.phrs.2021.105748

Figure Lengend Snippet: Fig. 3. ROS promoted accumulation of pyroptosis-related Tom20. a JC-1 kit was used to examine the MMP levels in A549 cells (Scale bar=25 µm). b, c ROS level was analyzed by flow cytometry in A549 cells (n ≥3, ***P＜0.001 vs CuB; ###P＜0.001 vs control group). d Effect of NAC on morphological feature changes in A549 cells (Scale bar=25 µm). e Western blotting analysis of the expression of GSDMD-FL, GSDMD-N, pro-IL-1β, mature IL-1β, pro-caspase-1 and cleaved caspase-1 in A549 cells. f Annexin V and 7-AAD double staining assay was used to identify the inhibition of NAC CuB-induced pyroptosis by NAC in A549 cells. g Immunofluorescence detection of the expression of mitochondrial protein Tom20 in A549 cells (Scale bar=10 µm). h The cell viability in Tom 20 knockdown A549 cells (n ≥3, ***P＜0.001 vs CuB; ###P＜0.001 vs si-Ctrl group).

Article Snippet: Antibodies for GSDMD (#93709), GAPDH (#8884), HMGB1 (#3935), cleaved-IL-1β (#83186), NLRP3 (#15101), cleaved-caspase1 (#4199), ASC (#13833), IL-1β (#12703), Gasdermin D (#93709), Tom20 (#42406), and the secondary antibodies including horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (#7074) were purchased from Cell Signaling Technology (CST, Danvers, MA, USA).

Techniques: Flow Cytometry, Control, Western Blot, Expressing, Double Staining, Inhibition, Immunofluorescence, Knockdown

Fig. 4. Cytosolic calcium accumulation was essential for CuB-induced pyroptosis. a, b Effect of BAPTA-AM (10 μM) or VX765 (60 µm) on Ca2+ levels in A549 cells (n ≥3, **P＜0.01, ***P＜0.001 vs CuB; ###P＜0.001 vs control group). c The process of the increase of Ca2+ levels in A549 cells was detected by using a Bio Tek CYTATION5 (Scale bar=25 µm). d Immunofluorescence assay was used to examine the expression of Ca2+ fluorescence (Scale bar=7.5 µm). e Effect of BAPTA-AM on the LDH release in A549 cells (n ≥3, ***P＜0.001 vs CuB; ###P＜0.001 vs control group). f Annexin V/7-AAD double staining assay was used to identify the inhibition of CuB-induced pyroptosis by BAPTA-AM in A549 cells. g The expression of GSDMD-FL, GSDMD-N, pro-IL-1β, mature IL-1β, pro-caspase-1 and cleaved caspase-1 in A549 cells.

Journal: Pharmacological research

Article Title: Cucurbitacin B inhibits non-small cell lung cancer in vivo and in vitro by triggering TLR4/NLRP3/GSDMD-dependent pyroptosis.

doi: 10.1016/j.phrs.2021.105748

Figure Lengend Snippet: Fig. 4. Cytosolic calcium accumulation was essential for CuB-induced pyroptosis. a, b Effect of BAPTA-AM (10 μM) or VX765 (60 µm) on Ca2+ levels in A549 cells (n ≥3, **P＜0.01, ***P＜0.001 vs CuB; ###P＜0.001 vs control group). c The process of the increase of Ca2+ levels in A549 cells was detected by using a Bio Tek CYTATION5 (Scale bar=25 µm). d Immunofluorescence assay was used to examine the expression of Ca2+ fluorescence (Scale bar=7.5 µm). e Effect of BAPTA-AM on the LDH release in A549 cells (n ≥3, ***P＜0.001 vs CuB; ###P＜0.001 vs control group). f Annexin V/7-AAD double staining assay was used to identify the inhibition of CuB-induced pyroptosis by BAPTA-AM in A549 cells. g The expression of GSDMD-FL, GSDMD-N, pro-IL-1β, mature IL-1β, pro-caspase-1 and cleaved caspase-1 in A549 cells.

Article Snippet: Antibodies for GSDMD (#93709), GAPDH (#8884), HMGB1 (#3935), cleaved-IL-1β (#83186), NLRP3 (#15101), cleaved-caspase1 (#4199), ASC (#13833), IL-1β (#12703), Gasdermin D (#93709), Tom20 (#42406), and the secondary antibodies including horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (#7074) were purchased from Cell Signaling Technology (CST, Danvers, MA, USA).

Techniques: Control, Immunofluorescence, Expressing, Fluorescence, Double Staining, Inhibition

Fig. 5. TLR4 activation promoted the CuB-induced pyroptosis. a The protein expression of TLR4 in A549 cells. b The molecular docking data of CuB on TLR4 dimer were analyzed by using autodock software. c CETSA-WB experiment to further confirm that CuB targeted TLR4 proteins. d The expression of GSDMD-FL, GSDMD-N, pro-IL-1β, mature IL-1β, pro-caspase-1, and cleaved caspase-1 in A549 cells. e Annexin V/7-AAD double staining assay was used to identify the inhi bition of CuB-induced pyroptosis by TAK242 in A549 cells. f The protein expression of TLR4 in A549 cells. g, h The cell viability and LDH activity in TLR4 knockdown A549 cells (n ≥3, *** P＜0.001 vs CuB; ###P＜0.001 vs NC group). i, j Mitochondrial ROS and cytosolic Ca2+ levels were detected by flow cytometry in A549 cells (n ≥3, ***P＜0.001 vs CuB; ###P＜0.001 vs NC group).

Journal: Pharmacological research

Article Title: Cucurbitacin B inhibits non-small cell lung cancer in vivo and in vitro by triggering TLR4/NLRP3/GSDMD-dependent pyroptosis.

doi: 10.1016/j.phrs.2021.105748

Figure Lengend Snippet: Fig. 5. TLR4 activation promoted the CuB-induced pyroptosis. a The protein expression of TLR4 in A549 cells. b The molecular docking data of CuB on TLR4 dimer were analyzed by using autodock software. c CETSA-WB experiment to further confirm that CuB targeted TLR4 proteins. d The expression of GSDMD-FL, GSDMD-N, pro-IL-1β, mature IL-1β, pro-caspase-1, and cleaved caspase-1 in A549 cells. e Annexin V/7-AAD double staining assay was used to identify the inhi bition of CuB-induced pyroptosis by TAK242 in A549 cells. f The protein expression of TLR4 in A549 cells. g, h The cell viability and LDH activity in TLR4 knockdown A549 cells (n ≥3, *** P＜0.001 vs CuB; ###P＜0.001 vs NC group). i, j Mitochondrial ROS and cytosolic Ca2+ levels were detected by flow cytometry in A549 cells (n ≥3, ***P＜0.001 vs CuB; ###P＜0.001 vs NC group).

Article Snippet: Antibodies for GSDMD (#93709), GAPDH (#8884), HMGB1 (#3935), cleaved-IL-1β (#83186), NLRP3 (#15101), cleaved-caspase1 (#4199), ASC (#13833), IL-1β (#12703), Gasdermin D (#93709), Tom20 (#42406), and the secondary antibodies including horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (#7074) were purchased from Cell Signaling Technology (CST, Danvers, MA, USA).

Techniques: Activation Assay, Expressing, Software, Double Staining, Activity Assay, Knockdown, Flow Cytometry

Fig. 7. CuB induced lung cancer cells pyroptosis in mouse models. a HE staining of heart, liver, spleen, lung, and kidney tissues in indicated groups (HE, original magnification, 200×, Scale bar=50 µm). b, c The mitochondrial ROS and cytosolic Ca2+ levels in tumor cells were detected by flow cytometry (n ≥3, ***P＜0.001 vs model group, ###P＜0.001 vs CuB 0.75 mg/kg group). d, e The protein expressions of TLR4, NEK7, NLRP3, ASC, Tom20, GSDMD-FL, GSDMD-N, pro-IL-1β, mature IL- 1β, pro-caspase-1, cleaved caspase-1and HMGB1 in tumor tissues were detected by western blotting.

Journal: Pharmacological research

Article Title: Cucurbitacin B inhibits non-small cell lung cancer in vivo and in vitro by triggering TLR4/NLRP3/GSDMD-dependent pyroptosis.

doi: 10.1016/j.phrs.2021.105748

Figure Lengend Snippet: Fig. 7. CuB induced lung cancer cells pyroptosis in mouse models. a HE staining of heart, liver, spleen, lung, and kidney tissues in indicated groups (HE, original magnification, 200×, Scale bar=50 µm). b, c The mitochondrial ROS and cytosolic Ca2+ levels in tumor cells were detected by flow cytometry (n ≥3, ***P＜0.001 vs model group, ###P＜0.001 vs CuB 0.75 mg/kg group). d, e The protein expressions of TLR4, NEK7, NLRP3, ASC, Tom20, GSDMD-FL, GSDMD-N, pro-IL-1β, mature IL- 1β, pro-caspase-1, cleaved caspase-1and HMGB1 in tumor tissues were detected by western blotting.

Article Snippet: Antibodies for GSDMD (#93709), GAPDH (#8884), HMGB1 (#3935), cleaved-IL-1β (#83186), NLRP3 (#15101), cleaved-caspase1 (#4199), ASC (#13833), IL-1β (#12703), Gasdermin D (#93709), Tom20 (#42406), and the secondary antibodies including horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (#7074) were purchased from Cell Signaling Technology (CST, Danvers, MA, USA).

Techniques: Staining, Flow Cytometry, Western Blot

Reduced cilium length is caused by enhanced γ-secretase activity and diminished Foxj1a levels. (a) 2.5-fold increased γ-secretase activity in Rer1p-depleted CL4 cells as indicated by increased NICD production ( n = 7). ctrl, control. (b) Overexpression of NICD but not of Cadherin ICD (CICD) reduces cilium length in CL4 cells. (c) Nanomolar concentrations of γ-secretase inhibition (InhX) rescue cilia length in Rer1p-depleted CL4 cells without affecting cilia length in control cells. Acetylated tubulin staining ( n = 150; three experiments). NSC, nonspecific control. (d) Reduced deltaA but unaffected notch1 and notch3 levels in 24-hpf SMO. (e) Enhanced Notch signaling in the ear sensory patch, but not in the tail, indicated by increased fluorescence of the Notch reporter line Tp1:hmgb1-mCherry. Quantification is shown of highlighted regions ( n > 15). UI, uninjected. (f) FM1-43 dye labeling shows a twofold reduction in the number of neuromasts in 3-dpf SMO. (g) Twofold reduction in the number of hair cells per neuromast (PLL) in 2-dpf SMO-injected ET4 transgenic embryos. Increased Notch signaling (100 pg NICD mRNA) results in less hair cells per neuromast (71% of control), whereas inhibiting Notch signaling (25 µM compound E [cpdE]) increases hair cell number (150% of control) and rescues the effect of SMO from 46 to 93.6% ( n > 12). (h) WISH shows normal differentiation of the DFC/KV lineage ( lrdr1 ) but reduced foxj1a levels in DFC (80% epiboly, arrows), KV (bud, arrows), pronephros (arrowheads), and notochord (asterisks). ss, somite stage. (i) Quantification of foxj1a levels by quantitative PCR in SMO. NICD does not affect foxj1a levels in the bud. NI, not injected. (j–l) Partial rescue of pronephros (j and k) and KV (l) cilia length upon coinjection of Foxj1a ( n ≥ 11 embryos; three experiments). Ac.tub, acetylated tubulin. Means ± SEM; *, P < 0.05; **, P < 0.01; ***, P < 0.001, Wilcoxon rank sum test (b and c) or Student’s t test (a, e, g, i, k, and l).

Journal: The Journal of Cell Biology

Article Title: Rer1p maintains ciliary length and signaling by regulating γ-secretase activity and Foxj1a levels

doi: 10.1083/jcb.201208175

Figure Lengend Snippet: Reduced cilium length is caused by enhanced γ-secretase activity and diminished Foxj1a levels. (a) 2.5-fold increased γ-secretase activity in Rer1p-depleted CL4 cells as indicated by increased NICD production ( n = 7). ctrl, control. (b) Overexpression of NICD but not of Cadherin ICD (CICD) reduces cilium length in CL4 cells. (c) Nanomolar concentrations of γ-secretase inhibition (InhX) rescue cilia length in Rer1p-depleted CL4 cells without affecting cilia length in control cells. Acetylated tubulin staining ( n = 150; three experiments). NSC, nonspecific control. (d) Reduced deltaA but unaffected notch1 and notch3 levels in 24-hpf SMO. (e) Enhanced Notch signaling in the ear sensory patch, but not in the tail, indicated by increased fluorescence of the Notch reporter line Tp1:hmgb1-mCherry. Quantification is shown of highlighted regions ( n > 15). UI, uninjected. (f) FM1-43 dye labeling shows a twofold reduction in the number of neuromasts in 3-dpf SMO. (g) Twofold reduction in the number of hair cells per neuromast (PLL) in 2-dpf SMO-injected ET4 transgenic embryos. Increased Notch signaling (100 pg NICD mRNA) results in less hair cells per neuromast (71% of control), whereas inhibiting Notch signaling (25 µM compound E [cpdE]) increases hair cell number (150% of control) and rescues the effect of SMO from 46 to 93.6% ( n > 12). (h) WISH shows normal differentiation of the DFC/KV lineage ( lrdr1 ) but reduced foxj1a levels in DFC (80% epiboly, arrows), KV (bud, arrows), pronephros (arrowheads), and notochord (asterisks). ss, somite stage. (i) Quantification of foxj1a levels by quantitative PCR in SMO. NICD does not affect foxj1a levels in the bud. NI, not injected. (j–l) Partial rescue of pronephros (j and k) and KV (l) cilia length upon coinjection of Foxj1a ( n ≥ 11 embryos; three experiments). Ac.tub, acetylated tubulin. Means ± SEM; *, P < 0.05; **, P < 0.01; ***, P < 0.001, Wilcoxon rank sum test (b and c) or Student’s t test (a, e, g, i, k, and l).

Article Snippet: Scales), Zpr1 and Zpr3 (Zebrafish International Resource Center, University of Oregon, Eugene, OR), c-Myc (A-14; sc789; Santa Cruz Biotechnology, Inc.), cleaved Notch1 (Val1744; Cell Signaling Technology), N-cadherin (BD), Ift20 (Abcam), and GMAP210 (BD), HA (3F10; Roche).

Techniques: Activity Assay, Over Expression, Inhibition, Staining, Fluorescence, Labeling, Injection, Transgenic Assay, Real-time Polymerase Chain Reaction

(A) Scheme of these experiments. (B, C) Example (B; scale bar, 100 µm) and quantification (C) of apoptotic cells found in the epidermis of mice of the indicated genotypes 24 h upon the application of DMBA ( n = 4). In (B), sections were stained with antibodies to the cleaved fragment of caspase 3 (ID number: 12367) and 14, 4′,6-diamidino-2-phenylindole (DAPI) to reveal apoptotic cells (red color) and cell nuclei (blue color), respectively. (D, E) Example (D; scale bar, 100 µm) and quantification (E) of phospho-histone H2AX + keratinocytes (brown color) present in the epidermis of short-term DMBA-treated mice of indicated genotypes ( n = 4). p-, phosphorylated. (F) Number of apoptotic (annexin V + ) wild-type and Vav2 −/− ; Vav3 −/− keratinocytes induced after 8 (left panel) and 12 (right panel) h in the indicated culture conditions ( n = 4). Bleo, bleomycin; DTT, dithiothreitol; NS, no statistically significant. (G, H) Example of a flow cytometry experiment (G) and subsequent quantification (H) of the level of apoptosis induced by either DMBA (G, H) or serum starvation (H) in wild-type and Vav2 −/− ; Vav3 −/− keratinocytes ectopically expressing GFP either alone (G, H) or in combination with HA-Vav2 (H) or Myc-Vav3 (G, H) ( n = 3). In (G), only the gated GFP + cells are shown.

Journal: PLoS Biology

Article Title: The Rho Exchange Factors Vav2 and Vav3 Favor Skin Tumor Initiation and Promotion by Engaging Extracellular Signaling Loops

doi: 10.1371/journal.pbio.1001615

Figure Lengend Snippet: (A) Scheme of these experiments. (B, C) Example (B; scale bar, 100 µm) and quantification (C) of apoptotic cells found in the epidermis of mice of the indicated genotypes 24 h upon the application of DMBA ( n = 4). In (B), sections were stained with antibodies to the cleaved fragment of caspase 3 (ID number: 12367) and 14, 4′,6-diamidino-2-phenylindole (DAPI) to reveal apoptotic cells (red color) and cell nuclei (blue color), respectively. (D, E) Example (D; scale bar, 100 µm) and quantification (E) of phospho-histone H2AX + keratinocytes (brown color) present in the epidermis of short-term DMBA-treated mice of indicated genotypes ( n = 4). p-, phosphorylated. (F) Number of apoptotic (annexin V + ) wild-type and Vav2 −/− ; Vav3 −/− keratinocytes induced after 8 (left panel) and 12 (right panel) h in the indicated culture conditions ( n = 4). Bleo, bleomycin; DTT, dithiothreitol; NS, no statistically significant. (G, H) Example of a flow cytometry experiment (G) and subsequent quantification (H) of the level of apoptosis induced by either DMBA (G, H) or serum starvation (H) in wild-type and Vav2 −/− ; Vav3 −/− keratinocytes ectopically expressing GFP either alone (G, H) or in combination with HA-Vav2 (H) or Myc-Vav3 (G, H) ( n = 3). In (G), only the gated GFP + cells are shown.

Article Snippet: In vitro cell cycle transitions were determined using the Click-iT EdU Alexa Fluor 647 Flow Cytometry Assay Kit (Invitrogen).

Techniques: Staining, Flow Cytometry, Expressing

(A) Quantification by flow cytometry of the number of EdU + , S-phase cells induced by the stimulation of quiescent keratinocytes of the indicated genotypes with either TPA or a synthetic keratinocyte growth media (CnT07). (B) Phosphorylation and expression status of Erk and Stat3 proteins in TPA-stimulated keratinocytes of indicated genotypes. (C) Rac1 (upper panel) and RhoA (lower panel) activation levels induced by the stimulation of quiescent keratinocytes of indicated genotypes with TPA ( n = 3). (D) Phosphorylation and expression status of Erk proteins in either serum- (two upper panels) or CnT07-stimulated (two bottom panels) keratinocytes of indicated genotypes. (E) Rac1 activation levels induced by the stimulation of quiescent keratinocytes with either serum (left panel) or CnT07 media (right panel) ( n = 3). (F) Immunoprecipitation experiments showing the tyrosine phosphorylation levels of endogenous Vav2 (top panel) and ectopically expressed HA-Vav2 (third panel from top) in wild-type keratinocytes treated with TPA (+) in the absence (−) or the presence (+) of the indicated drugs ( n = 2). Due to problems with the detection of the endogenous proteins after blot stripping, the loading control shown in the second panel from top was made using a parallel immunoprecipitation with the same anti-Vav2 antibody. GF, GF109203X; IP, immunoprecipitation. (G) Western blot of total cellular extracts showing the levels of phosphorylation (top) and expression (bottom) of Erk proteins in wild-type keratinocytes stimulated as indicated in (F) ( n = 3). (H) Rac1 activation levels induced by TPA in wild-type keratinocytes under the indicated experimental conditions ( n = 3). Go, Gö 6976; sh Fyn , cells infected with a Fyn-specific shRNA.

Journal: PLoS Biology

Article Title: The Rho Exchange Factors Vav2 and Vav3 Favor Skin Tumor Initiation and Promotion by Engaging Extracellular Signaling Loops

doi: 10.1371/journal.pbio.1001615

Figure Lengend Snippet: (A) Quantification by flow cytometry of the number of EdU + , S-phase cells induced by the stimulation of quiescent keratinocytes of the indicated genotypes with either TPA or a synthetic keratinocyte growth media (CnT07). (B) Phosphorylation and expression status of Erk and Stat3 proteins in TPA-stimulated keratinocytes of indicated genotypes. (C) Rac1 (upper panel) and RhoA (lower panel) activation levels induced by the stimulation of quiescent keratinocytes of indicated genotypes with TPA ( n = 3). (D) Phosphorylation and expression status of Erk proteins in either serum- (two upper panels) or CnT07-stimulated (two bottom panels) keratinocytes of indicated genotypes. (E) Rac1 activation levels induced by the stimulation of quiescent keratinocytes with either serum (left panel) or CnT07 media (right panel) ( n = 3). (F) Immunoprecipitation experiments showing the tyrosine phosphorylation levels of endogenous Vav2 (top panel) and ectopically expressed HA-Vav2 (third panel from top) in wild-type keratinocytes treated with TPA (+) in the absence (−) or the presence (+) of the indicated drugs ( n = 2). Due to problems with the detection of the endogenous proteins after blot stripping, the loading control shown in the second panel from top was made using a parallel immunoprecipitation with the same anti-Vav2 antibody. GF, GF109203X; IP, immunoprecipitation. (G) Western blot of total cellular extracts showing the levels of phosphorylation (top) and expression (bottom) of Erk proteins in wild-type keratinocytes stimulated as indicated in (F) ( n = 3). (H) Rac1 activation levels induced by TPA in wild-type keratinocytes under the indicated experimental conditions ( n = 3). Go, Gö 6976; sh Fyn , cells infected with a Fyn-specific shRNA.

Article Snippet: In vitro cell cycle transitions were determined using the Click-iT EdU Alexa Fluor 647 Flow Cytometry Assay Kit (Invitrogen).

Techniques: Flow Cytometry, Expressing, Activation Assay, Immunoprecipitation, Stripping, Western Blot, Infection, shRNA

(A) Quantification by flow cytometry of the apoptosis induced in pure (No) and genotypically mixed (Yes) cultures of wild-type and Vav2 −/− ; Vav3 −/− keratinocytes upon 12 h in complete media with DMBA or under serum-free media conditions. (B) Quantification by flow cytometry of the apoptosis observed in wild-type and Vav2 −/− ; Vav3 −/− keratinocytes kept for 12 h with serum, without serum, or without serum plus the indicated extracellular factors ( n = 3). (C) Determination by flow cytometry of the percentage of wild-type and Vav2 −/− ; Vav3 −/− keratinocytes that have entered S phase after 4.5 h under the indicated culture conditions ( n = 3). (D–F) Phosphorylation and expression status of Erk and Stat3 in serum-starved wild-type and Vav2 −/− ; Vav3 −/− keratinocytes stimulated with either EGF (D) or IL6 (E–G) for the indicated periods of time. In panels (F) and (G), keratinocytes were transduced with empty and Vav-encoding lentiviruses prior to the starvation and stimulation steps as indicated in the respective panel ( n = 3). (H) Tyrosine phosphorylation levels of endogenous Vav2 (top panel) in wild-type keratinocytes treated with the indicated agents for 5 min ( n = 2). Due to problems with the detection of the endogenous proteins after blot stripping, the loading control shown in the second panel from top was made using a parallel immunoprecipitation with the same anti-Vav2 antibody.

Journal: PLoS Biology

Article Title: The Rho Exchange Factors Vav2 and Vav3 Favor Skin Tumor Initiation and Promotion by Engaging Extracellular Signaling Loops

doi: 10.1371/journal.pbio.1001615

Figure Lengend Snippet: (A) Quantification by flow cytometry of the apoptosis induced in pure (No) and genotypically mixed (Yes) cultures of wild-type and Vav2 −/− ; Vav3 −/− keratinocytes upon 12 h in complete media with DMBA or under serum-free media conditions. (B) Quantification by flow cytometry of the apoptosis observed in wild-type and Vav2 −/− ; Vav3 −/− keratinocytes kept for 12 h with serum, without serum, or without serum plus the indicated extracellular factors ( n = 3). (C) Determination by flow cytometry of the percentage of wild-type and Vav2 −/− ; Vav3 −/− keratinocytes that have entered S phase after 4.5 h under the indicated culture conditions ( n = 3). (D–F) Phosphorylation and expression status of Erk and Stat3 in serum-starved wild-type and Vav2 −/− ; Vav3 −/− keratinocytes stimulated with either EGF (D) or IL6 (E–G) for the indicated periods of time. In panels (F) and (G), keratinocytes were transduced with empty and Vav-encoding lentiviruses prior to the starvation and stimulation steps as indicated in the respective panel ( n = 3). (H) Tyrosine phosphorylation levels of endogenous Vav2 (top panel) in wild-type keratinocytes treated with the indicated agents for 5 min ( n = 2). Due to problems with the detection of the endogenous proteins after blot stripping, the loading control shown in the second panel from top was made using a parallel immunoprecipitation with the same anti-Vav2 antibody.

Article Snippet: In vitro cell cycle transitions were determined using the Click-iT EdU Alexa Fluor 647 Flow Cytometry Assay Kit (Invitrogen).

Techniques: Flow Cytometry, Expressing, Transduction, Stripping, Immunoprecipitation