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发布于:2018-8-8 18:58:05  访问:108 次 回复:0 篇
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Consequences [24]. Recently we reported that higher glucose (HG) situation reduces the
For higher glucose (HG) situation, low glucose culture medium was replaced with DMEM containing 4.5 g/L D-glucose (Life Technology-Invitrogen) supplemented with 2 FBS for 24 h, as Decernotinib site previously reported [25?8], before performing other treatment options for the indicated instances. To generate a cell line stably expressing GFP-LC3, three ?105 RKO cells have been cultured into 6-well plates and transfected with 5 g of pEGFP-LC3 expression vector (kindly provided by Moshe Oren, Weizmann Institute of Science, Rehovot, Israel) with Lipofectamine Plus (Invitrogen, Monza, Italy) in line with the manufacturer‘s guidelines. Forty-eight hours soon after transfection cells underwent selection with geneticin G418 (1.five mg/ml). Following 10?four days, the selected GFP-LC3positive clones have been visualized on a Nikon Eclipse Ti-U fluorescence microscope (Nikon). Chemotherapeutic drug Adriamycin (ADR) (Sigma) was added in culture medium at two g/ml for 16?four h; the inhibitor of autophagic protein degradation chloroquine (CQ) [29] (Sigma-Aldrich) was added in culture medium at 25 M for 16 h; p53 inhibitor pifithryn- (PFT-) [30] (ENZO Life Sciences, Lausen Switzerland) was used at 30 M for 16?four h, as reported [31].Viability assayFor viability assay, subconfluent cells have been plated in duplicate in 60 mm multiwell Petri dishes and 24 h PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/27663262 later culture medium was replaced with HG or low glucose medium, each containing 2 FBS. The day after, ADR (2 g/ml) have been added to cell cultures for 16?4 PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/27766426 h. Both floating and adherent cells were collected and cell viability was determined by Trypan blue (Sigma, St. Louis, MO,Garufi et al. Journal of Experimental Clinical Cancer Study (2017) 36:Web page three ofUSA) exclusion by direct counting having a haemocytometer. The percentage of cell death, as blue/total cells, was assayed by scoring about 200 cells per well in triplicate.Cell death/PI stainingRNA extraction and semi-quantitative reverse transcription (RT)-PCR analysisCell death was quantified by Fluorescence Activated Cell Sorting (FACS) analysis, staining cells together with the nonvital dye propidium iodide (PI) (Immunological Sciences, Rome, Italy), following the manufacturer‘s instruction [32].Consequences [24]. Recently we reported that high glucose (HG) situation reduces the tumor cell response to druginduced apoptosis due to impairment of p53 apoptotic function. Mechanistically we identified that HG induces homeodomain-interacting protein kinase two (HIPK2)protein degradation leading to impairment of HIPK2/ p53 apoptotic axis with inhibition of p53 apoptotic targets PUMA and p53AIP1 [25, 26]. In this study, we present evidence that HG condition modifications the p53 transcriptional activity from PUMA to DRAM together with the consequence of impairment of drug-induced cell death. We also show that p53-induced DRAM sustains HG-triggered autophagy and that either DRAM or autophagy inhibition restore both PUMA transcription and drug-induced cell death in vitro and in vivo.MethodsCell culture and reagentsIn this study colon cancer RKO, HCT116, and HCT116p53-/- cells have been utilized. Cells have been routinely cultured in DMEM (Dulbecco‘s Modified Eagle‘s medium) (Life Technology-Invitrogen, Eggenstein, Germany) containing 1 g/L D-glucose (low glucose - LG), supplemented with 10 heat-inactivated fetal bovine serum (FBS) plus one hundred units/ml penicillin/streptomycin, and glutamine, in 5 CO2 humidified incubator at 37 .
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