RNA sequencing analysis of isoginkgetin and thapsigargin treated HCT116 and HCT116 ATF4 deficient cells
Activating transcription factor 4 (ATF4) is activated during cellular stress through a pathway called the integrated stress response (ISR). We had previously reported that the splicing inhibitor isoginkgetin (IGG) activates ATF4 and ATF4-dependent transcripts. To determine the role of ATF4 in the transcriptional response to IGG, we used tandem CRISPR cas9 gene editing to create an ATF4 deficient HCT116 (colon cancer) cell line. We completed RNA sequencing on HCT116 parental and HCT116 ATF4 deficient cells treated with IGG, and thapsigargin (Tg), a positive control for ATF4 activation. We found that IGG led to the differential expression of 76 transcripts, and 58 of these were dependent on ATF4. Tg led to a far more robust transcriptional response, which appeared to be less ATF4 dependent. Overall design: Total RNA was collected from mock-treated, vehicle control-treated (DMSO), IGG (30µM) and Tg (2µM) drug treated HCT116 and HCT116 ATF4 deficient cells following an 8h exposure, of a 2h vehicle control (DMSO) and 2h Tg (2µM) treatment. Four biological experiments were included in both cell lines. RNA was quantified using the Qubit 4 Fluorometer with the Qubit RNA Broad range Assay kit. RNA quality was determined using the 4200 TapeStation System. Library prep was completed following the Illumina stranded mRNA prep reference guide, and pooled samples were loaded into a NextSeq 2000 Reagent Cartridge and sequenced using an Illumina NextSeq 2000. The data was analyzed using the Omics data analysis framework for regulatory application (R-ODAF) pipeline (https://github.com/R-ODAF/R-ODAF_Health_Canada/) and differentially expressed genes were identified using the DESeq2 package.