Fig. 1

The LNCaP–iSnail model is a dynamic model of epithelial–mesenchymal plasticity. a Western blot showing protein expression for Snail and E-cadherin in untreated LNCaP–iSnail cells, and cells treated with Dox for 1 and 3 days. γ-Tubulin was visualised for loading control purposes. b Gene expression of E-cadherin and vimentin LNCaP–iSnail cells treated with Dox for 1 and 3 days. Gene expression was normalised to RPL32. Fold change is relative to the respective LNCaP–iGFP cells treated with Dox for 1 and 3 days. One-way ANOVA, *p < 0.05. Error bars indicate SEM for biological triplicates. c Representative immunofluorescence images showing expression of Snail, vimentin, and E-cadherin in untreated LNCaP–iSnail cells and treated with Dox for 5 days. Nuclei were visualised with DAPI. Scale bars = 50 µm. d Phase contrast images of LNCaP–iSnail cells grown as multicellular spheroids in a Matrigel™ assay prior to treatment (Day 0) and treated with Dox for 2 and 4 days. Scale bars = 100 µm. e Heatmap showing the expression of known epithelial and mesenchymal genes from untreated LNCaP–iSnail and LNCaP–iGFP spheroids, and treated with Dox for 2 and 4 days. Heatmap color refers to normalised z-score. f GSEA enrichment plots of established EMT signatures [14, 20] within the transcriptional program regulated by 4 days of Snail-induction, compared to Dox-treated LNCaP–iGFP cells. g Western blot showing Snail protein levels in untreated parental LNCaP cells, untreated LNCaP–iSnail cells, and LNCaP–iSnail cells treated with Dox for 7 days followed by removal for 7 days. GAPDH protein was visualised for loading control purposes. h Western blot showing expression of Snail, vimentin, E-cadherin, and EpCAM proteins in untreated LNCaP–iSnail cells, and treated with Dox for 1 and 3 days followed by removal for 3, 5, 10, and 20 days. γ-Tubulin was visualised for loading control purposes. i Gene expression of Snail, vimentin, Zeb1, E-cadherin, EpCAM, ESRP1, and ESRP2 in LNCaP–iSnail cells treated with Dox for 3 and 5 days, followed by removal for 3, 5, and 20 days. Gene expression was normalised to RPL32. Fold change is relative to untreated cells. One-way ANOVA, *p < 0.05. Error bars indicate SEM for biological triplicates. j Representative immunofluorescence images of E-cadherin and vimentin proteins in untreated LNCaP–iSnail cells, and treated with dox for 5 days followed by removal for 7 days. Nuclei were visualised using DAPI. Scale bars = 50 µm