cell apoptosis dapi detection kit  (Keygen Biotech)

Journal: International Journal of Molecular Sciences

Article Title: p38 Regulates FoxO3a-Mediated SOD2 Expression to Prevent Cd-Induced Oxidative Stress in Neuronal Cells

doi: 10.3390/ijms262210919

Figure Lengend Snippet: Cd induces the nuclear expression of FoxO3a in neuronal cells. SH-SY5Y cells were treated with 5 μM Cd for various time periods. ( A ) The cell lysates were subjected to Western Blot analysis to measure FoxO3a protein expression; ( B ) nuclear and cytoplasmic extracts were subjected to Western Blot analysis to measure FoxO3a expression. ( C , D ) SH-SY5Y cells and rat cerebral cortical neurons were exposed to 5 μM Cd for 6 h, and confocal laser scanning microscopy was performed to determine FoxO3a localization. FoxO3a is shown in green, and nuclei were counterstained with DAPI (blue). All results are representative of three independent experiments. The provided scale bar in the merged image represents 20 μm. * p < 0.05, ** p < 0.01 versus the control group.

Article Snippet: The cells were incubated with DAPI (Cell Apoptosis DAPI Detection Kit, KGA215, KeyGEN BioTECH, Nanjing, China) according to the manufacturer’s instructions.

Techniques: Expressing, Western Blot, Confocal Laser Scanning Microscopy, Control

Journal: International Journal of Molecular Sciences

Article Title: p38 Regulates FoxO3a-Mediated SOD2 Expression to Prevent Cd-Induced Oxidative Stress in Neuronal Cells

doi: 10.3390/ijms262210919

Figure Lengend Snippet: p38 regulates the Cd-induced nuclear expression of FoxO3a. ( A ) SH-SY5Y cells were treated with 5 μM Cd for various time periods, and phosphorylated and total MAPK proteins were detected by Western Blot analysis. ( B ) SH-SY5Y cells were treated with 5 μM Cd for 6 h after preincubating with 10 μM SB203580 for 1 h. Western Blot analysis was performed to determine FoxO3a expression. ( C , D ) SH-SY5Y cells and rat cerebral cortical neurons were exposed to 5 μM Cd for 6 h after preincubation with 10 μM SB203580 for 1 h. Confocal laser scanning microscopy was performed to determine FoxO3a expression. FoxO3a is shown in green, and nuclei were counterstained with DAPI (blue). ( E ) SH-SY5Y cells were treated with 5 μM Cd for various time periods. Western Blot analysis was performed to determine phosphorylation of FoxO3a-Ser7. ( F – H ) SH-SY5Y cells and rat cerebral cortical neurons were incubated with 5 μM Cd for 6 h after preincubation with 10 μM SB203580 for 1 h. Western Blot analysis and confocal laser scanning microscopy were performed to determine phosphorylation of FoxO3a-Ser7 expression. The provided scale bar in the merged image represents 20 μm. ( G – H ) p-FoxO3a is shown in red, and nuclei were counterstained with DAPI (blue). All results are representative of three independent experiments. * p < 0.05, ** p < 0.01 versus the control group; # p < 0.05, ## p < 0.01 versus the Cd group.

Article Snippet: The cells were incubated with DAPI (Cell Apoptosis DAPI Detection Kit, KGA215, KeyGEN BioTECH, Nanjing, China) according to the manufacturer’s instructions.

Techniques: Expressing, Western Blot, Confocal Laser Scanning Microscopy, Phospho-proteomics, Incubation, Control

Journal: Journal of Translational Medicine

Article Title: A novel small molecule glycolysis inhibitor WZ35 exerts anti-cancer effect via metabolic reprogramming

doi: 10.1186/s12967-022-03758-0

Figure Lengend Snippet: WZ35 can inhibit YAP from entering the nucleus. A HCCLM3 cell line was transfected with shRNA or pcDNA/peGFP vectors and harvested 48 h post-transfection to quantify the protein level of YAP and GAPDH. B and C Colony formation assay of different cell lines showed significant changes in stably transfected cells with representative images being shown and colony numbers being quantified. D CCK-8 assays were performed after cell lines were transfected with YAP shRNA or pcDNA/peGFP vectors. E The structure and synthesis of WZ35. F Western blotting analysis of the protein level of YAP, Cyr61 and CTGF in HCCLM3 cells treated with DMSO, curcumin and WZ35. G Western blot analysis of subcellular distribution of YAP in HCCLM3 cells treated with DMSO and WZ35.Histone H3 and Tublin are markers for nuclear and cytoplasm, respectively. Nuc., nucleus; Cyt., cytoplasm. H Representative immunofluorescent images via confocal microscopy were displayed with bars of 10 μm, confirming the reduction of nuclear localization of YAP protein. (Green: YAP; Blue: DAPI)

Article Snippet: Cell Apoptosis DAPI Detection Kit, DCFH-DA ROS detection kit, and pEGFP-hYAP 1 were obtained from Addgene (Shanghai, China).

Techniques: Transfection, shRNA, Colony Assay, Stable Transfection, CCK-8 Assay, Western Blot, Confocal Microscopy

Journal: Journal of Translational Medicine

Article Title: A novel small molecule glycolysis inhibitor WZ35 exerts anti-cancer effect via metabolic reprogramming

doi: 10.1186/s12967-022-03758-0

Figure Lengend Snippet: WZ35 exhibits significant antitumor activity. A CCK-8 assays were utilized to measure the suppression rate of curcumin and WZ35 (with the concentration of 0, 5, 10, 20 to 40 μg/mL) treated HCCLM3, HepG2, and Huh7 cells. B Colony formation assays of HCCLM3, HepG2, and Huh7 cells were conducted by plating 5000 indicated cells per well in 6-well plates to measure the effect of curcumin and WZ35 on clonogenicity with representative images and quantification of colony numbers being shown. C Representative fluorescence microscopy displaying nuclear transformations in HCCLM3, HepG2, and Huh7 cells following 18 h treatment of WZ35 (10 μg/mL) or treatment of curcumin (10 μg/mL) by means of DAPI staining. Results are presented as the mean ± standard error from independent experiments performed in triplicate. * P < 0.05, ** P < 0.01, student’s t test. D The levels of various apoptosis associated proteins, including Bax, Bcl-2, and Cleaved caspase-3 were detected by western blotting following the 18 h treatment of WZ35, with GAPDH serving as an internal control. E Quantitation of the murine body weight and tumor volumes over time in four indicated tumor xenograft groups, as measured by utilizing electronic balance and caliper. The results are presented as the mean ± standard error from independent experiments in triplicate. * P < 0.05, ** P < 0.01, *** P < 0.001, student’s t test. F Subcutaneous tumor xenograft models of WZ35-treated HCCLM3 were used. Mice were put to death by cervical dislocation and the tumors were harvested after 17 days. Optical image of each tumor and the weight curve was photographed and quantified, respectively. (n = 6 mice per group). G ) The representative images of Hematoxylin and eosin staining of indicated treated tumors have been shown. Scale bars, 200 μm for images

Article Snippet: Cell Apoptosis DAPI Detection Kit, DCFH-DA ROS detection kit, and pEGFP-hYAP 1 were obtained from Addgene (Shanghai, China).

Techniques: Activity Assay, CCK-8 Assay, Concentration Assay, Fluorescence, Microscopy, Staining, Western Blot, Control, Quantitation Assay

Journal: Journal of Translational Medicine

Article Title: A novel small molecule glycolysis inhibitor WZ35 exerts anti-cancer effect via metabolic reprogramming

doi: 10.1186/s12967-022-03758-0

Figure Lengend Snippet: WZ35 inhibits growth of liver cancer cells in a ROS-dependent manner. A The levels of intracellular reactive oxygen species (ROS) were determined by measuring the mean fluorescence intensity (MFI) of DCFH-DA (DCF) via flow cytometry in control or WZ35-treated HCCLM3 cells following pretreatment with NAC. Results are presented as the mean ± standard error from independent experiments in triplicate. *** P < 0.001, student’s t test. B – D HCCLM3 cells with or without NAC (5 mM) pretreatment were incubated with or without WZ35 (10 μg/mL) to assess the cellular proliferation via CCK-8 assays B and colony formation assays C , assess cellular apoptosis via DAPI staining assays D with representative images and/or quantifications have been shown. E Western blotting analysis of the protein level of YAP and CTGF in HCCLM3 cells treated with NAC and WZ35. All these results are presented as the mean ± standard error from independent experiments in triplicate. ** P < 0.01, *** P < 0.001, student’s t test

Article Snippet: Cell Apoptosis DAPI Detection Kit, DCFH-DA ROS detection kit, and pEGFP-hYAP 1 were obtained from Addgene (Shanghai, China).

Techniques: Fluorescence, Flow Cytometry, Control, Incubation, CCK-8 Assay, Staining, Western Blot

Journal: Journal of Translational Medicine

Article Title: A novel small molecule glycolysis inhibitor WZ35 exerts anti-cancer effect via metabolic reprogramming

doi: 10.1186/s12967-022-03758-0

Figure Lengend Snippet: WZ35 antitumor activity depends upon inhibition of the YAP protein and allows it to regulate the expression of GLUT1. A – C DAPI staining assays A , colony formation assays B and CCK-8 assays C were conducted to measure apoptosis and cellular viability in control or WZ35-treated (10 μg/mL) HCCLM3 cells with or without the pretreatment of knockdown or overexpression via shRNA or pcDNA/peGFP vectors. D Barchart visualized significantly related genes in central carbon metabolism with higher YAP1 expression including 10 genes with the highest logFC and 4 genes with the lowest logFC from datasets GSE97098, GSE77314 and GSE153783. E Correlation between YAP1 and SLC2A1 in liver cancer samples from TIMER dataset ( P = 9.94 × 10 –11 ) was visualized in a scatter diagram. F Western blotting analysis of the YAP and GLUT1 protein level of HCCLM3 cells treated with WZ35 and plasmid. G Real-time qPCR analysis of the YAP and GLUT1 mRNA level of HCCLM3 cells treated with WZ35 and plasmid. H Predicted TEAD binding motif site sequence in the promoter region of GLUT1 host gene chromosome 1 from the database JASPAR

Article Snippet: Cell Apoptosis DAPI Detection Kit, DCFH-DA ROS detection kit, and pEGFP-hYAP 1 were obtained from Addgene (Shanghai, China).

Techniques: Activity Assay, Inhibition, Expressing, Staining, CCK-8 Assay, Control, Knockdown, Over Expression, shRNA, Western Blot, Plasmid Preparation, Binding Assay, Sequencing