Dissecting primary (translation independent) from secondary (translation dependent) IFN-mediated differential gene expression

NIH-3T3 cells were pretreated for 15 min with either DMSO (mock) or cycloheximide followed by addition of either mock, 100 U/ml IFNalpha or 100 U/ml IFNgamma for 1h. During the last 30 min, 500 µM 4-thiouridine was added to cell culture medium. Total cellular RNA was isolated using Trizol reagent and nascent RNA was purified as described (Dölken et al. RNA 2008) . Three replicates of nascent RNA were analyzed by Affymetrix Mouse Gene ST 1.0 arrays Primary (translation independent) from secondary (translation dependent) IFN-mediated differential gene expression were studied in NIH-3T3 fibroblasts by studying studying differential gene expression in presence and absence of Cycloheximide (CHX). In addition, the effect of 75 min CHX during the last 30 min of treatment was studied in nascent RNA.

将NIH-3T3细胞以二甲基亚砜（DMSO，空白对照）或环己酰亚胺（cycloheximide, CHX）预处理15分钟，随后分别加入空白对照、100 U/ml干扰素α（IFNα）或100 U/ml干扰素γ（IFNγ）处理1小时。在上述处理的最后30分钟，向细胞培养基中添加500 μM 4-硫尿苷（4-thiouridine）。
使用Trizol试剂（Trizol reagent）提取总细胞RNA，并参照Dölken等人2008年发表于《RNA》期刊的方法纯化新生RNA（nascent RNA）。通过Affymetrix小鼠基因ST 1.0芯片（Affymetrix Mouse Gene ST 1.0 arrays）对3份新生RNA重复样本进行检测分析。
本研究以NIH-3T3成纤维细胞为模型，通过对比存在与缺失环己酰亚胺条件下的基因表达差异，探究了干扰素介导的差异基因表达中不依赖翻译的初级效应与依赖翻译的次级效应；此外，本研究还针对新生RNA样本，探究了在处理的最后30分钟施加75分钟环己酰亚胺处理所产生的影响。