protease inhibitor cocktail set iii  (Millipore)

Journal: Cancer Research and Treatment : Official Journal of Korean Cancer Association

Article Title: The Histone Deacetylase Inhibitor Entinostat Mediates HER2 Downregulation in Gastric Cancer, Providing the Basis for Its Particular Efficacy in HER2 -Amplified Tumors and in Combination Therapies

doi: 10.4143/crt.2024.546

Figure Lengend Snippet: Entinostat effect on microRNA levels and microRNA-dependent regulation of human epidermal growth factor receptor 2 (HER2) expression. (A) Schematic illustration of miRNAs regulating HER2 and/or affected by HER2. (B) Alterations in miRNA expression levels in NCI-N87 cells after entinostat treatment for 48 hours with the indicated concentrations. (C) Time-dependent reduction of HER2 protein levels upon transfection of NCI-N87 cells with 10 nM of miR-205-3p. (D) Effect of anti-miR transfection on HER2 expression upon entinostat treatment. HER2 levels after transfection of control miRNA (ctrl) were compared with HER2 levels after transfection of specific miR-205 inhibitor (205). For the protein analyses, HER2 levels were monitored 72 hours after miR (C) or anti-miR (D) transfection. Mean±standard error of mean of three independent experiments are given. a) p < 0.05.

Article Snippet: The next day, cells were treated for 72 hours, prior to harvesting and lysis in 20-50 μL RIPA buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate) containing protease inhibitor cocktail set III, EDTA-free (1:500, Merck Darmstadt).

Techniques: Expressing, Transfection, Control

Journal: Cancer Research and Treatment : Official Journal of Korean Cancer Association

Article Title: The Histone Deacetylase Inhibitor Entinostat Mediates HER2 Downregulation in Gastric Cancer, Providing the Basis for Its Particular Efficacy in HER2 -Amplified Tumors and in Combination Therapies

doi: 10.4143/crt.2024.546

Figure Lengend Snippet: Effect of combination therapy with entinostat plus selective human epidermal growth factor receptor 2 (HER2) inhibitor CP724714. (A) Analysis of HER2 protein expression in MKN-74 cells treated for 72 hours with vehicle dimethyl sulfoxide (DMSO, D), 1.5 μM entinostat (E), 2.0 μM CP-724714 (CP), or the combination of both (E+CP). Densitometric quantifications of three independent experiments (mean±standard error of mean [SEM]) and one representative western blot are shown. (B) Effect of entinostat, CP724714, or the combination of both on proliferation of MKN-74 cells in comparison to vehicle-treated cells as determined by Cell Counting Kit 8 (CCK8) assay (mean±SEM, n=3). (C) Effect on colony formation of MKN-74 after treatment with entinostat, CP, or entinostat+CP. Scale bars=500 µm. (D) Combinatory effect on cell death, analysis was conducted using flow cytometry-based distribution of propidium iodide (PI) and annexin-V–stained cells. (E) Combinatory effect on cell cycle, analysis was conducted using flow cytometry–based cell cycle distribution of PI-stained cells. Quantitation of three independent experiments (mean±SEM) are shown in (D) and (E). a) p < 0.05, b) p < 0.03, c) p < 0.01.

Article Snippet: The next day, cells were treated for 72 hours, prior to harvesting and lysis in 20-50 μL RIPA buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate) containing protease inhibitor cocktail set III, EDTA-free (1:500, Merck Darmstadt).

Techniques: Expressing, Western Blot, Comparison, Cell Counting, CCK-8 Assay, Flow Cytometry, Staining, Cell Cycle Assay, Quantitation Assay