Figure 3

Verification of selected gene and protein expression. Verification of selected gene and protein expression was performed using RT-qPCR and direct ALDH enzyme-staining assays as described in Methods. (a) RT-qPCR was used to verify overexpression of selected genes identified by RNA-Seq analyses. The blue, pink, and green bars represent 3 separate experiments each consisting of 3 replicate samples for each primer set. One-way ANOVA analyses indicate the mean value increases in gene expression between E and R cells in all experiments for TENS4 (p < 0.03), ALDH3A1 (p < 0.01), CLDN-2 (p < 0.01) and AKR1B10 (P < 0.005) represent statistically significant differences. (b-c) fluorometric ALDH enzyme assays were used to directly measure the relative expression of ALDH in R and E cells in cell monolayers undergoing E-R transition on soft substrates. 90% of R cells, as well as some E cells in the cell islands undergoing E-R transition express relatively high amounts of ALDH activity. Scale bar: 100 μm. (d) The integrated cellular fluorescence intensity of ALDH for R cells was 4–5 fold higher as compared to E cells.