Journal: International journal of molecular sciences

Article Title: Regulation of the Golgi Apparatus by p38 and JNK Kinases during Cellular Stress Responses.

doi: 10.3390/ijms22179595

Figure Lengend Snippet: Figure 1. Phospho-proteomic analysis of p38- and JNK-dependent phosphorylation reactions. (a). Schematic of phospho- proteomic workflow. U2OS cells were treated in duplicates with indicated combinations of anisomycin (Ani, 1 h) and p38 and JNK inhibitors (p38i, JNKi, 0.5 h pre-treatment). Lysates were digested with trypsin and peptides were labeled with

Article Snippet: Membranes were blocked in PBS-T + 5% milk and then probed with the following antibodies overnight: Phospho RxxS*/T* (Cell signaling, 9614, rabbit), GIGYF1 (Abcam, ab121784 and ab121646, rabbit), MK2 (Cell signaling, 3042, rabbit), CHK1 (S345, Cell signaling, 2358, rabbit), 14–3–3 (pan, Cell signaling, 8312, rabbit), GST (Santa Cruz, discontinued, sc459, rabbit), TTP (Sigma, T5327, rabbit), ZNF598 (Abcam, ab111698, rabbit and/or Sigma, HPA041760, rabbit), FLAG (Sigma, F1804, rabbit and/or Sigma, F1804, mouse) or 4EHP (Cell signaling, 6916, rabbit), GRASP55 (Abcam, Ab211532, mouse), CAMSAP2 (Novus, Abingdon, UK, NCP1–21402, rabbit), TJAP1 (Sigma, HPA030165, rabbit), GM130 (BD biosciences, Franklin Lakes, NJ, USA Clone 35, mouse), YIPF2 (Santa Cruz, sc-398530, mouse), p-JNK (Cell signaling, 9255, mouse), p-p38 (T180/Y182, Cell signaling, 9216, mouse), p150 (BD biosciences, 610473, mouse), MCM6 (Santa Cruz, goat), HA (Santa Cruz, sc-7392, mouse) and GFP (Torrey Pines, Secaucus, NJ, USA TP401, rabbit).

Techniques: Phospho-proteomics, Labeling

Journal: International journal of molecular sciences

Article Title: Regulation of the Golgi Apparatus by p38 and JNK Kinases during Cellular Stress Responses.

doi: 10.3390/ijms22179595

Figure Lengend Snippet: Figure 3. GIGYF1 phospho-mutant retains p-body localization upon cellular stress. (a). U2OS cells were transfected with the indicated siRNA, pre-treated with p38 and MK2 inhibitors (0.5 h) and UV-irradiated (50 J/m2, 1 h recovery) as indicated. Lysates were incubated with recombinant GST-14–3–3 protein or GST alone. GST pull-down material (PD: GST) and whole

Article Snippet: Membranes were blocked in PBS-T + 5% milk and then probed with the following antibodies overnight: Phospho RxxS*/T* (Cell signaling, 9614, rabbit), GIGYF1 (Abcam, ab121784 and ab121646, rabbit), MK2 (Cell signaling, 3042, rabbit), CHK1 (S345, Cell signaling, 2358, rabbit), 14–3–3 (pan, Cell signaling, 8312, rabbit), GST (Santa Cruz, discontinued, sc459, rabbit), TTP (Sigma, T5327, rabbit), ZNF598 (Abcam, ab111698, rabbit and/or Sigma, HPA041760, rabbit), FLAG (Sigma, F1804, rabbit and/or Sigma, F1804, mouse) or 4EHP (Cell signaling, 6916, rabbit), GRASP55 (Abcam, Ab211532, mouse), CAMSAP2 (Novus, Abingdon, UK, NCP1–21402, rabbit), TJAP1 (Sigma, HPA030165, rabbit), GM130 (BD biosciences, Franklin Lakes, NJ, USA Clone 35, mouse), YIPF2 (Santa Cruz, sc-398530, mouse), p-JNK (Cell signaling, 9255, mouse), p-p38 (T180/Y182, Cell signaling, 9216, mouse), p150 (BD biosciences, 610473, mouse), MCM6 (Santa Cruz, goat), HA (Santa Cruz, sc-7392, mouse) and GFP (Torrey Pines, Secaucus, NJ, USA TP401, rabbit).

Techniques: Mutagenesis, Transfection, Irradiation, Incubation, Recombinant

Journal: International journal of molecular sciences

Article Title: Regulation of the Golgi Apparatus by p38 and JNK Kinases during Cellular Stress Responses.

doi: 10.3390/ijms22179595

Figure Lengend Snippet: Figure 4. JNK phosphorylation targets in the Golgi apparatus. (a). JNK- and p38-dependent phosphorylation sites on Golgi apparatus-resident proteins or Golgi trafficking proteins extracted from Figure 1c, Table S1 and [11]. (b). U2OS cells were pre-treated with JNK and p38 inhibitors (JNKi, p38i, 0.5 h) and treated with anisomycin (Ani, 1 h) as indicated. Lysates were separated by SDS-PAGE or phos-tag gel and analyzed by immunoblotting with the indicated antibodies.

Article Snippet: Membranes were blocked in PBS-T + 5% milk and then probed with the following antibodies overnight: Phospho RxxS*/T* (Cell signaling, 9614, rabbit), GIGYF1 (Abcam, ab121784 and ab121646, rabbit), MK2 (Cell signaling, 3042, rabbit), CHK1 (S345, Cell signaling, 2358, rabbit), 14–3–3 (pan, Cell signaling, 8312, rabbit), GST (Santa Cruz, discontinued, sc459, rabbit), TTP (Sigma, T5327, rabbit), ZNF598 (Abcam, ab111698, rabbit and/or Sigma, HPA041760, rabbit), FLAG (Sigma, F1804, rabbit and/or Sigma, F1804, mouse) or 4EHP (Cell signaling, 6916, rabbit), GRASP55 (Abcam, Ab211532, mouse), CAMSAP2 (Novus, Abingdon, UK, NCP1–21402, rabbit), TJAP1 (Sigma, HPA030165, rabbit), GM130 (BD biosciences, Franklin Lakes, NJ, USA Clone 35, mouse), YIPF2 (Santa Cruz, sc-398530, mouse), p-JNK (Cell signaling, 9255, mouse), p-p38 (T180/Y182, Cell signaling, 9216, mouse), p150 (BD biosciences, 610473, mouse), MCM6 (Santa Cruz, goat), HA (Santa Cruz, sc-7392, mouse) and GFP (Torrey Pines, Secaucus, NJ, USA TP401, rabbit).

Techniques: Phospho-proteomics, SDS Page, Western Blot

Journal: International journal of molecular sciences

Article Title: Regulation of the Golgi Apparatus by p38 and JNK Kinases during Cellular Stress Responses.

doi: 10.3390/ijms22179595

Figure Lengend Snippet: Figure 5. Regulation of Golgi morphology by p38 and JNK. (a). Heatmap and horizontal clustering of descriptors of Golgi morphology. U2OS cells were pre-treated with the combination of JNK and p38 inhibitors (JNKi, p38i, 0.5 h) and treated with anisomycin (Ani, 1 h) or IL1β (1 h) as indicated. Cells were fixed, immunostained with antibodies against cis Golgi markers GM130, GRASP65 and/or trans Golgi markers GRASP55 and TGN46 and images were acquired by high content microscopy. Images were processed and analyzed with CellProfiler software for calculation of the indicated parameters, and are presented as log2-transformed mean fold changes compared to the control. n > 1700 cells. (b). Box plots of selected

Article Snippet: Membranes were blocked in PBS-T + 5% milk and then probed with the following antibodies overnight: Phospho RxxS*/T* (Cell signaling, 9614, rabbit), GIGYF1 (Abcam, ab121784 and ab121646, rabbit), MK2 (Cell signaling, 3042, rabbit), CHK1 (S345, Cell signaling, 2358, rabbit), 14–3–3 (pan, Cell signaling, 8312, rabbit), GST (Santa Cruz, discontinued, sc459, rabbit), TTP (Sigma, T5327, rabbit), ZNF598 (Abcam, ab111698, rabbit and/or Sigma, HPA041760, rabbit), FLAG (Sigma, F1804, rabbit and/or Sigma, F1804, mouse) or 4EHP (Cell signaling, 6916, rabbit), GRASP55 (Abcam, Ab211532, mouse), CAMSAP2 (Novus, Abingdon, UK, NCP1–21402, rabbit), TJAP1 (Sigma, HPA030165, rabbit), GM130 (BD biosciences, Franklin Lakes, NJ, USA Clone 35, mouse), YIPF2 (Santa Cruz, sc-398530, mouse), p-JNK (Cell signaling, 9255, mouse), p-p38 (T180/Y182, Cell signaling, 9216, mouse), p150 (BD biosciences, 610473, mouse), MCM6 (Santa Cruz, goat), HA (Santa Cruz, sc-7392, mouse) and GFP (Torrey Pines, Secaucus, NJ, USA TP401, rabbit).

Techniques: Microscopy, Software, Transformation Assay, Control

Journal: Scientific reports

Article Title: AEG-1 silencing attenuates M2-polarization of glioma-associated microglia/macrophages and sensitizes glioma cells to temozolomide.

doi: 10.1038/s41598-021-96647-3

Figure Lengend Snippet: Figure 6. AEG-1 activates Wnt/β-catenin signaling via targeting GSK-3β in glioma cells. (A) Enriched top 10 KEGG pathways for Affymetrix microarray of AEG-1 NC (N = 3) and KD (N = 3) glioma cells. 22 DEGs were enriched in KEGG_WNT_SIGNALING PATHWAY. P-value = 1.02E−06. [drawn by R 3.6.0 (https://cran.r- project.org/doc/FAQ/R-FAQ.html#Citing-R)]. (B) Enrichment plot of the Wnt signaling pathway from GSEA; ‘h’ and ‘l’ represented AEG-1 high and low expression, respectively. NES = 1.523, NOM P-value = 0.008, FDR q-value = 0.216 [drawn by GSEA tool (version 4.1.0)]. (C) Western blot bands of AEG-1, β-catenin, GSK-3β, cyclin D1, and CD44 in NC and shAEG-1 glioma cell lines. (D) Relative protein abundance was calculated by ImageJ and GraphPad Prism 8.2.1 software. (E, F) In U251 and U87 cells, Co-IP assays showed the direct interaction of AEG-1 and GSK-3β. (G) Immunofluorescence assays were used to detect the localization of AEG-1 (red) and GSK-3β (green) in U251 and U87 cells. All data were presented as the mean ± SD from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: Cells were incubated with rabbit anti-AEG-1 antibody (Proteintech, 1:1000), mouse anti-GSK-3β antibody (Cell Signaling Technology, 1:1000), and rabbit anti-γH2AX (Cell Signaling Technology, 1:400), diluted using 1% FBS, at 4 °C overnight.

Techniques: Microarray, Expressing, Western Blot, Quantitative Proteomics, Software, Co-Immunoprecipitation Assay, Immunofluorescence

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: 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: International journal of biological sciences

Article Title: Specific Pyruvate Kinase M2 Inhibitor, Compound 3K, Induces Autophagic Cell Death through Disruption of the Glycolysis Pathway in Ovarian Cancer Cells.

doi: 10.7150/ijbs.59855

Figure Lengend Snippet: Figure 4. Compound 3K reduced colony formation and cell proliferation and induced the cell cycle arrest in SK-OV-3 cells. (A) Effect of compound 3K (1, 2.5, or 5 µM) on proliferation of SK-OV-3 cells following treatment for 48 h. Cell proliferation was checked with an IncuCyte software every 6 h. (B) Representative photographs and quantitative analysis of colony formation assay of SK-OV-3 cells treated with indicated compound 3K concentrations in 6-well plates. Colony areas were measured using image analyzer. (C) SK-OV-3 cells were treated with compound 3K (1, 2.5, or 5 µM) for 24 h. After treatment, the cells were stained with propidium iodide (PI) and analyzed using flow cytometry. (D) Bar graph indicating the different phases of cell cycle distributions of SK-OV-3 cells treated with 0.1% DMSO control or compound 3K. (E) Effect of compound 3K on the expression of cell cycle-related proteins. SK-OV-3 cells were treated for 24 h with compound 3K (1, 2.5, or 5 µM). The expression of cyclin B1, p-Cdc2, and Cdc2 were analyzed using western blot analysis. The values represent the mean ± SD. *p < 0.05 and **p < 0.01.

Article Snippet: Primary antibodies against PKM2, PKM1, Cyclin B1, p-Cdc2, Cdc2, Bax, Bcl-2, caspase 3, Beclin-1, LC3B, Atg5, Atg7, p62, MMP-2, MMP-9, TIMP-2, p-AKT, AKT, p-AMPK, AMPK, p-mTOR, mTOR, p-p70S6K, p70S6K, GLUT1, LDHA, MCT4, and β-actin were all obtained from Cell Signaling (Beverly, MA, USA).

Techniques: Software, Colony Assay, Staining, Flow Cytometry, Control, Expressing, Western Blot

Journal: Frontiers in endocrinology

Article Title: Transcriptional Analysis of Sepsis-Induced Activation and Damage of the Adrenal Endothelial Microvascular Cells.

doi: 10.3389/fendo.2019.00944

Figure Lengend Snippet: FIGURE 3 | mRNA and protein verification of RNA sequencing analysis. Real-time PCR (qPCR) verification of expression of endothelial-specific genes (A) and expression of some of the top 100 most altered genes (B) with specific primers. Relative gene expression was determined by the 11Ct method with normalization of 18S ribosomal RNA. Saline-treated samples were depicted with open bars, and LPS-treated samples were depicted with black filled bars. N = 5 and statistics were calculated with the two-tailed non-parametric Mann-Whitney test. Values are mean with SEM. *p < 0.05, **p < 0.01. Images of fluorescent staining of thrombomodulin (Thmd) and Vascular adhesion molecule 1 (VCAM-1) (C) in adrenal tissue sections of mouse adrenal gland isolated from either Saline or LPS treated mice. Thmd and VCAM-1 were visualized using secondary antibodies coupled to Cy3 dye (red color). Western blot evaluation of VCAM-1 expression in adrenal glands isolated from mice treated either with Saline or LPS (N = 4) (D). Graphic representation of the VCAM-1 and GAPDH protein expression quantified in ImageJ Software based on the densitometric evaluation of protein bands (E). Results and evaluation of dot-blot based protein array (F) of different inflammatory-related molecules (G), cytokines (H) and chemokines (I). Saline-treated samples were depicted with open bars, and LPS-treated samples were depicted with gray filled bars. For each experimental group, two adrenals from three different animals (N = 6) were pooled and results are presented as double replicate.

Article Snippet: For the VCAM-1 and GAPDH detection following antibodies were used rabbit monoclonal anti-mouse VCAM-1 antibody (1:1000, clone EPR5047, ab134047, Abcam), a monoclonal rabbit anti-mouse GAPDH (1:1000; clone 14C10, #2118, Cell Signaling Technology), and goat anti-rabbit HRP conjugated secondary antibodies (1:6000, CST).

Techniques: RNA Sequencing, Real-time Polymerase Chain Reaction, Expressing, Gene Expression, Saline, Two Tailed Test, MANN-WHITNEY, Staining, Isolation, Western Blot, Software, Dot Blot, Protein Array