RGS2-mediated translational control drives cellular dormancy and resistance to ER-stress-induced apoptosis by inducing proteasome-mediated ATF4 degradation

Slow-cycling/dormant cancer cells (SCCs) are thought to cause cancer relapse, but the underlying biology remains obscure. A subpopulation of SCCs has been found even in rapidly growing tumors and cancer cell lines. To investigate the mechanism underlying cellular dormancy, we obtained SCCs from three NSCLC cell lines (H460, H1299, and SK-MES-1) and PDX tumor by using a cell proliferation-dependent fluorescent dye (CSFE). Overall design: Three non-small cell lung cancer (NSCLC) cell lines (H460, H1299 and SK-MES-1) and two primary cell lines derived from different parts of NSCLC PDX tissue (PDX1-1, PDX1-2) were labelled with Carboxyfluorescein succinimidyl ester (CFSE) and then cultured for 7 days to distinguish active-cycling cells (ACCs, CFSElow) and dye remained slow-cycling cells (SCCs, CFSEhigh). Cultured cells were sorted into CFSEhigh (upper, >90%) and CFSElow (lower, <10%) population by flow cytometry. Sorted cells were washed twice with PBS and then RNA was extracted using Trizol reagent (Qiagen). 500 ng of total RNA was used for the construction of sequencing libraries. RNA libraries were prepared for sequencing using LEXOGEN Quant-Seq Library Prep Kit (Cat#001.24) with standard protocols. For deata process, Illumina Casava1.8 software used for basecalling. Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence(using fastx_trimmer), then mapped to hg19 whole genome using Bowtie2. Read count extraction and nomalization were performed using edgeR.