Transcriptome analysis of resistant colon adenocarcinoma cells after treatment with symmetric selenoesters. undefined

Doxorubicin resistant Colo 320/MDR-LRP (ATCC-CCL-220.1) cell line expressing ABCB1, was purchased from LGC Promochem (Teddington, UK). The cells were cultured in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), 2 mM L-glutamine, 1 mM Na-pyruvate, 100 mM Hepes, nystatin and a penicillin-streptomycin mixture in concentrations of 100 U/L and 10 mg/L, respectively. The density of the cells was adjusted to 5105 cells in 1 ml of RPMI medium and the cells were transferred in 1 ml aliquots into a 24-well plate. After an overnight incubation the cells were treated with different selenoesters1 using a non-toxic concentration based on their respective IC50. The samples were prepared in triplicates using an untreated control and selenoesters as follows: - methylketone selenoester EDAG-1 and EDAG-5 were applied at 0.25 µM - methyloxycarbonyl selenoester EDAG-6 was applied at 1 µM - cyano-selenoesters EDAG-10 and EDAG-11 were applied at 0.5 µM After the incubation of 24 h in a humidified atmosphere (5% CO2, 95% air) at 37 ºC the cells were removed from the 24-well-plate using a cell scraper. Then, RNA isolation and cDNA library preparation and whole transcriptome sequencing was performed by DeltaBio 2000 Kft. (Szeged, Hungary). The libraries were pooled and mixed with 1% PhiX DNA and sequenced on Illumina NextSeq 550 sequencing platform with NextSeq 500/550 High Output Kit v2.5 in 75 cycles (Illumina).

表达ATP结合盒转运蛋白B1（ABCB1）的阿霉素耐药Colo 320/MDR-LRP（ATCC-CCL-220.1）细胞系购自英国特丁顿的LGC Promochem公司。将细胞培养于添加了10%热灭活胎牛血清（FBS）、2 mM L-谷氨酰胺、1 mM丙酮酸钠、100 mM HEPES、制霉菌素，以及终浓度分别为100 U/L和10 mg/L的青霉素-链霉素混合液的RPMI-1640培养基中。将细胞密度调整为每1 mL RPMI培养基中含5×10⁵个细胞，以1 mL每孔的体积接种至24孔培养板。过夜培养后，根据各硒酯类化合物1对应的半数抑制浓度（IC50）选用无毒浓度，使用不同的硒酯类化合物处理细胞。
样本设置3个生物学重复，包含未处理对照组及如下硒酯类化合物处理组：
- 甲基酮类硒酯EDAG-1与EDAG-5，给药浓度为0.25 µM
- 甲氧羰基类硒酯EDAG-6，给药浓度为1 µM
- 氰基硒酯类EDAG-10与EDAG-11，给药浓度为0.5 µM
将细胞置于37 ℃、含5% CO₂的湿润空气培养箱中孵育24 h后，使用细胞刮刮取24孔培养板中的细胞。随后由匈牙利塞格德的DeltaBio 2000 Kft.完成RNA提取、cDNA文库制备及全转录组测序。将各文库混合并添加1%的PhiX DNA，随后使用Illumina NextSeq 550测序平台，搭配NextSeq 500/550 High Output Kit v2.5，以75个循环完成测序。