Microarray analysis of isoginkgetin-treated HCT116 and HCT116p53-/- cells

Isoginkgetin (IGG) is a small molecule inhibitor of the spliceosome although the direct target remains elusive. Widespread failure to accurately remove introns poses a risk to cells and organisms through the potential production of aberrant mRNAs and proteins. The cellular responses to accumulation of these intermediates and/or the direct interference of spliceosome assembly itself may constitute a splicing stress but this is not well defined yet. We used oligonucleotide microarrays to assess genome wide changes in gene expression associated with exposure to IGG in HCT116 cells and an isogenic subline lacking the p53 tumor suppressor that responds to a variety of transcriptional stresses (Oncogene 18 (3), 583-592). Two of the 3 enriched pathways identified using PANTHER analysis of differentially expressed transcripts are linked to the ATF4 transcription factor and these effects are p53-independent. Total RNA was collected from mock-treated, vehicle control-treated (DMSO) and drug-treated (30μM IGG) HCT116 cells and a p53 null subline, following an 8 hour continuous exposure. Two independent biological experiments were performed with each cell line. An Agilent Bioanalyzer was used to ensure RNA integrity before labeling and hybridizing to Affymetrix Human Transcriptome 2.0 oligonucleotide microarrays at the Affymetrix Microarray Facility, Stemcore Laboratory, Ottawa Hospital Research Institute (Ottawa, ON, Canada). Affymetrix Transcriptome Analysis Console (TAC) 4.0 was used with default settings. A one-way between-subject unpaired ANOVA was used to determine statistical significance and was subject to false discovery rate (FDR) multi-test correction (Benjamini-Hochberg Step-Up FDR).