Glucose uptake measurements in PIK3CA-mutant ovarian cancer cell lines

Description

This dataset contains glucose uptake measurements (via 2-NBDG assay) in PIK3CA-mutant ovarian cancer cell lines (OVCAR-3 and A2780) . Data files include: - Original datas - Ethical review materials

Steps to reproduce

Patient Tissue Samples Reagents and antibodies Animal studies Cell lines The A2780 and OVCAR3 cell lines were purchased from the Cell Bank of the Chinese Academy of Sciences. The cells were maintained in 37 °C supplemented with 10% FBS and 1% penicillin / streptomycin (streptomycin 100 μ g / mL, penicillin 100U/mL) 1640 medium, and 5% carbon dioxide in the humidified incubator. Cellular glycolytic stress experiments Cell mitochondrial pressure assay Untargeted mass spectrometry detection Western blotting Cell proliferation assays The proliferation capacity of the different cells was assessed by CCK8 assay. Cells were seeded into 96-well plates (2×104/well), at the indicated time-points, the old medium was replaced with 100 μL of fresh medium containing 10 μL of CCK8 solution. After 4 hours of incubation at 37°C, the absorbance levels for each sample at 540 nm were measured using a Multiskan Ascent plate reader (Thermo Electron, New York, NY, USA). Cell viability assay The effect of SFN or MK2206 on the viability of cells was detected by MTT assay. Cells were seeded into 96-well plates (6×104/well) and treated with various concentrations of SFN (0,1.25,2.5, 5, 10, 20, 40 and 80 μM). At the indicated time-points, the old medium was replaced with 100 μL of fresh medium containing 20 μL of MTT solution (5 mg/mL in PBS). After 4 hours of incubation at 37°C, formazan crystals were completely solubilized with 150 μL DMSO in each well. The absorbance levels for each sample at 540 nm were measured. The data were duplicated three times at least. Overexpression lentivirus transfection According to the full-length cDNA sequence of the human PIK3CA gene in Gene Bank, with the vector (CMV-MCS-3flag-polyA-EF1A-zsGreen-sv 40-puromycin) as the skeleton, was constructed For the PIK3CA-E545K mutation and wild-type virus, the virus packaging was completed by Shanghai Jikai Gene Company. Statistical methods GraphPad Prism 8.0.1 was used for statistical analysis and statistical graphing. Measurement information was used to describe the trend of centralized dispersion using mean (x) plus or minus standard deviation (s). The t-test or paired t-test was used to compare the differences in means between two groups, and the analysis of variance (ANOVA) or repeated-measures ANOVA was used to test the differences in means between multiple groups. The Kaplan-Meier method was used for survival analysis, and the log-rank test was used to compare the differences between groups. All statistical tests were two-sided and a P value less than 0.05 will be considered statistically significant. P-values <0.05, <0.01, <0.001, and <0.0001 are respectively marked as "*", " * *", "* * *", "* * * *".

Institutions

• Zhengzhou University

  Henan, Zhengzhou

Categories

Funders

• National Youth Science Foundation

  West Virginia, United States

  Grant ID: 81802770
• Beijing Science and Technology Innovation Medical Development Foundation

  Grant ID: KC2021-JX-0186-130
• CSCO-Zai Ding cancer treatment research fund project

  Grant ID: Y-zai2022/ms-0246
• Henan Provincial Health and Health Commission, Joint Co-construction Project of Medical Science and Technology Tackling Program in Henan Province

  Grant ID: 2018020056
• Natural Science Foundation of Henan

  Grant ID: 242300421273

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