• Thu. Sep 21st, 2023

A working solution was prepared by mixing the calibrator, distilled water, and cell samples, and suspended in a water bath at 37 C for 15 minutes


Oct 3, 2021

A working solution was prepared by mixing the calibrator, distilled water, and cell samples, and suspended in a water bath at 37 C for 15 minutes. of molecular cell biology and experimental zoology (establishing hyperglycemic nude mouse model for colon cancer transplantation by BALB/C nude mice) to find the effects of on proliferation, migration, and metabolism of colon cancer cells and in colon cancer and hyperglycemia by providing a theoretical and experimental basis for developing a target gene Ct ? internal Igfbp3 reference Ct (mean standard deviation); Ct = Ct of the target gene in the sample to be tested ? Ct of the target gene in the reference sample (mean standard deviation; if there is no reference sample, select the sample with the most extensive Ct as the reference for calculation). The relative sample initial template amount 2?Ct (mean standard deviation). The results were analyzed using the ABI Prism 7300 SDS Software (Applied Biosystems Foster City, CA, USA), and the relative RNA expression was calculated. The experiment was repeated three times. Table PAT-048 1 shRNA target sequence of gene was detected by qPCR. The procedure followed for qPCR is the same that was mention in section Results. Construction of CCAT-1 lentivirus vectors to transfect colon cancer cells and cell grouping The DNA oligonucleotide was designed with the software designer 3.0 (GENEPHARMA, CHN), and GENEPHARMA Pharmaceutical Technology Co. provided the synthetic primers, Ltd. Reagents included DNA endonuclease (BpiI, #ER1011, PstI, #ER0611, BamHI, #ER0051, MBI Fermentas, Lithuania), DNA ligase (#EL0011, MBI Fermentas, Lithuania), DNA markers (#SM0161, MBI Fermentas, Lithuania). For the construction of lentivirus vectors, shRNA primers were synthesized and connected to the U6 promoter of lentivirus vectors for constructing the target shRNA plasmid. Following this, shRNA plasmid and the auxiliary plasmid were co-transfected into 293T cells for verification, and the computer virus solution was collected, concentrated, and decided. The colon cancer cell line SW620 (ATCC CCL-228, USA) was infected with shRNA plasmid computer virus solution. RNAi interference efficiency was identified via qPCR. Based on silencing, vacant vector transfection, and different glucose medium cultivating environments, the cells were divided into four groups: for 10 minutes, and the culture medium was discarded. The cell suspension was rinsed three times with precooled PBS; an adequate amount of precooled PBS was added to resuspend the cells, and the cells were lysed by ultrasound exposure. The centrifugation was performed at 4 C for PAT-048 10 minutes at 10,000 g, cell fragments were removed, and the supernatant was assimilated. The procedures were performed according to the steps included in the Glucose ELISA Kit (YZB/Hu 2624-40-2014, Shanghai Rongsheng Biology, CHN) and Lactic acid ELISA Kit (A019-2, Nanjing Jiancheng, CHN). A working solution was prepared by mixing the calibrator, distilled water, and cell samples, and suspended in a water bath at 37 C for 15 minutes. The absorbance values at PAT-048 505 nm (glucose) and 530 nm (lactic acid) were separately decided using an automatic biochemical analyzer (TBA-40, TOSHIBA, JPN). Considering the standard substances concentration as the abscissa and the OD value as the ordinate, a standard curve was drawn. The sample calculation formula is usually: sample concentration (mmol/L) = (measured OD value ? blank OD value)/(standard OD value ? blank OD value) standard concentration (mmol/L) dilution occasions before test. Protein expression detected by Western blotting The expression levels of LDH-A, pkm2, hK2, Bcl-2, Bax, E-cadherin, vimentin, phosphatidylinositol-3-kinases (PI3K)/protein serine-threonine kinase (Akt)/cellular-myelocytomatosis viral oncogene (C-MYC), and their phosphorylated proteins were detected by Western blotting. The total protein was extracted using the Ripa lysate and measured via the Bradford method. Following this, 50 g of total protein was used for gel electrophoresis (300 mA), and the proteins were transferred onto a PVDF membrane (Millipore, IPVH00010, USA). The transfer efficiency was decided via Coomassie brilliant blue staining. The PVDF film was rinsed for 5 minutes with 25 mL Tris-buffered saline (TBS), and 5% degreasing milk powder with TBS was added as a sealing solution. The solution was then placed on an orbital shaker (MaxQ8000, Thermo Scientific, USA) to shake gently overnight at 4 C; further, 25 mL TBS was used to scour the membrane three times. The PVDF membrane was dipped into 3 mL of diluted corresponding antibodies: LDH (dilution ratio 1:5,000, 300 mA, 35 minutes), PKM2 (dilution ratio 1:1,000,.