Gene expression profiling of GSK3 inhibitor (CHIR99021)-treated intestinal organoids

Acting downstream of many growth factors, extracellular signal-regulated kinase (ERK) plays a pivotal role in regulating cell proliferation and tumorigenesis, where its spatiotemporal dynamics, as well as its strength, determine cellular responses. Here, we uncover the ERK activity dynamics in intestinal epithelial cells (IECs) and their association with tumour characteristics. In vivo imaging identified two distinct modes of ERK activity, sustained and pulse-like activity, in IECs. The sustained and pulse-like activity depended on ErbB2 and EGFR, respectively. Notably, deregulated activation of Wnt signalling, the earliest event in intestinal tumorigenesis, augmented EGFR signalling and exalted it to a dominant driver of ERK activity dynamics, which rendered IECs addicted to EGFR signalling. Furthermore, the frequency of ERK activity pulses was also increased to promote cell proliferation. Thus, ERK activity dynamics are defined by composite inputs from EGFR and ErbB2 signalling in IECs and their alterations underlie tumour-specific sensitivity to pharmacological EGFR inhibition. In this microarray analysis, we aimed to elucidate molecular mechanisms that mediate Wnt signalling activation-induced alterations in EGFR-ERK signalling dynamics. For microarray analysis, we performed two independent series of experiments. Total RNA was extracted from intestinal organoids cultured in the ENR media supplemented with or without CHIR99021 (5 microM) for four days as described above. Synthesis of cDNA, in vitro transcription and labelling of cRNA, and hybridization to the Mouse Gene 1.0 ST arrays (Affymetrix, Santa Clara, CA, USA) were performed according to the manufactures’ protocols. The CEL files were analyzed by Transcriptome Analysis Console (TAC) 4.0 software (Thermo Fisher Scientific). Expression signals of all genes (probe sets) were calculated using the RMA algorithm.