Figure 5
Transcriptomic analysis of CD44hi/CD24lo G4 and CD44hi/CD24− A4 clones identifying genes and gene signatures related to the CSC-like phenotype and potential new biomarkers. (A) Heat map of the genes differentially expressed more than two-fold in A4 relative to G4 (FDR 0.01), showing representative gene clusters. (B) Heat map focusing on gene expression levels of EMT markers: VIM, transcription factors (ZEB1, ZEB2, TWIST1), snail 1 and 2 (SNAI1/SNAI2), matrix metalloproteinase-2 (MMP2), E-cadherin (CDH1) and claudins (CLDN3, CLDN4, CLDN7) and CSC markers: CD24, CD44, aldehyde dehydrogenase (ALDH1A1), epithelial cell adhesion (ESA) molecule, MUC1, integrin β1 (ITGB1) and integrin α6 (ITGA6). Each colored square represents the relative transcript abundance (log 2 space) of each of five biological replicates of A4 and G4, respectively. Highest expression is red and lowest is blue. (C) Flow cytometry analysis of LSR and RAB25 showing higher expression in A4 versus G4. Controls without primary antibody were included. (D) Comparison of the expression levels of ZEB1 and LSR in G4, A4 and HMT3909S13 by ICC showing higher expression of ZEB1 and lower expression of LSR in G4 versus A4. Scale bar: 20 µm. (E) Western blotting of ZEB1 and S100A14 showing higher expression of ZEB1 and lower expression of S100A14 in G4 versus A4. The parental cell line (HMT3909S13) shows intermediate expression of both proteins. See also Supplementary Figure S4 and Supplementary Table S2.