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. 1. CuB induced non-apoptotic cell death. a The chemical structure of CuB. b, c Effect of CuB on cell viability and LDH activity in A549 cells (n ≥3, **P＜0.01, ***P＜0.001 vs control group). d The caspase-3, −7, −8, and −9 proteins expression in CuB treatment A549 cells for 24 h. e The caspase-3/−7 activity in A549 cells after CuB treatment for 24 h (n ≥3). f Inhibitory effect of z-Vad-fmk on the cell viability in CuB- treated A549 cells (n ≥3, ns＞0.05 vs CuB; ##P＜0.01, ###P＜0.001 vs control group). g Flow cytometry analysis of Annexin V and 7AAD double staining. h PI uptake progress and the fluorescence value in A549 cells after CuB (100 nM) treatment was detected by Bio Tek CYTATION5 (Scale bar=25 µm).

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: Activity Assay, Control, Expressing, Flow Cytometry, Double Staining, Fluorescence

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

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

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

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

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

Journal: Frontiers in endocrinology

Article Title: Excessive Iodine Promotes Pyroptosis of Thyroid Follicular Epithelial Cells in Hashimoto's Thyroiditis Through the ROS-NF-κB-NLRP3 Pathway.

doi: 10.3389/fendo.2019.00778

Figure Lengend Snippet: FIGURE 1 | Aberrant pyroptosis occurs in the thyroid tissues of HT patients and is induced by excessive iodine in vitro. (A,B) Gasdermin D (GSDMD) protein levels in the thyroid tissue of HT patients (n = 20) and controls (n = 10) were analyzed by immunoblots. “Control” indicates tissues from patients with nodular goiter of the thyroid. Representative immunoblotting results and quantification of GSDMD are shown. (C,D) Representative results of GSDMD immunohistochemical staining in HT tissues (n = 20) and control tissues (n = 10) are shown. Brown regions represent positive expression (original magnification, ×200; scale bars, 100 µm). (E–G) Nthy-ori3-1 cells were harvested after treatment with a gradient of concentrations of sodium iodide (NaI) for 24 h. The images presented are immunoblots probed for GSDMD (Hsp60 served as the loading control). All statistical results shown are representative of three replicates. (H) The cell viability of Nthy-ori 3-1 cells was assessed by CCK-8 assays after NaI treatment for 24 h. All statistical results shown are representative of three replicates. Significant differences and P-values were calculated by unpaired t-tests or one-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Thyroid sections were blocked with 2% bovine serum albumin in PBS for 30min and then incubated with mouse anti-human GSDMD or mouse antihuman IL-1β antibodies (Santa Cruz, NJ, USA) at 4◦C overnight.

Techniques: In Vitro, Western Blot, Control, Immunohistochemical staining, Staining, Expressing, CCK-8 Assay

Journal: Frontiers in endocrinology

Article Title: Excessive Iodine Promotes Pyroptosis of Thyroid Follicular Epithelial Cells in Hashimoto's Thyroiditis Through the ROS-NF-κB-NLRP3 Pathway.

doi: 10.3389/fendo.2019.00778

Figure Lengend Snippet: FIGURE 3 | The NLRP3 inflammasome participates in excessive iodine-induced pyroptosis in TFCs. (A,B) The NLRP3 expression levels were measured by immunoblots when Nthy-ori 3-1 cells were treated with NaI with or without NAC (10 mM) or IKK-16 (2 µM) for 24 h. (C,D) Verification of the silencing efficiency of NLRP3 by siRNA in Nthy-ori3-1 cells is shown by immunoblots; NC indicates the negative control. (E,F) The protein levels of GSDMD were detected by immunoblots after transfection of siNLRP3 in NaI-treated Nthy-ori 3-1 cells. All statistical results shown are representative of three replicates. Significant differences and P-values were calculated by one-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Thyroid sections were blocked with 2% bovine serum albumin in PBS for 30min and then incubated with mouse anti-human GSDMD or mouse antihuman IL-1β antibodies (Santa Cruz, NJ, USA) at 4◦C overnight.

Techniques: Expressing, Western Blot, Negative Control, Transfection

Journal: Frontiers in endocrinology

Article Title: Excessive Iodine Promotes Pyroptosis of Thyroid Follicular Epithelial Cells in Hashimoto's Thyroiditis Through the ROS-NF-κB-NLRP3 Pathway.

doi: 10.3389/fendo.2019.00778

Figure Lengend Snippet: FIGURE 5 | Proposed model of TFC pyroptosis in excessive iodine-promoted HT. Excessive iodine enters TFCs and induces the production of ROS, which activates NF-κB signaling and increases GSDMD-FL. In addition, the NLRP3 inflammasome is activated by NF-κB signaling, leading to GSDMD-FL cleavage into GSDMD-N, which triggers pore formation in the membrane and the release of mature IL-1β from cells, causing a sterile inflammatory response and further contributing to pyroptotic cell death and the subsequent promotion of HT.

Article Snippet: Thyroid sections were blocked with 2% bovine serum albumin in PBS for 30min and then incubated with mouse anti-human GSDMD or mouse antihuman IL-1β antibodies (Santa Cruz, NJ, USA) at 4◦C overnight.

Techniques: Membrane, Sterility