Fig. 3

Snail-regulated EMP influences cell cycle and metabolic activity in prostate cancer cells. a Enrichment of indicated hallmark datasets from the MSigDB [14] during the EMT/MErT timecourse. b Heatmap showing the expression of selected cell cycle-related genes in untreated LNCaP–iSnail cells (No Dox), cells treated with dox for 5 days (EMT5; E5), and subsequently removed for 3, 5, and 20 days (MErT3-20). Heatmap color refers to normalised z-score. c Gene expression of CCNB1, CDKN3, FOXM1, and MKI67 in LNCaP–iSnail cells treated with dox for 5 days (EMT5), and subsequently removed for 3, 5, and 20 days (MErT3-20). Gene expression was normalised to RPL32. Fold change is relative to untreated LNCaP–iSnail cells (NoDox). One-way ANOVA, *p < 0.05. Error bars indicate SEM for biological triplicates. d Flow cytometry cell cycle analysis of untreated LNCaP–iSnail and LNCaP–iGFP cells, and cells treated with dox for 5 days (EMT5) and subsequently removed for 5 and 10 days (MErT5–10). One-way ANOVA, *p < 0.05. Error bars indicate SEM for biological triplicates. e Histogram representation of flow cytometry analysis for the expression of Ki-67 antigen surface expression in untreated LNCaP–iSnail cells (D0), and cells treated with dox for 5 days (EMT5), followed by removal for 5 days (MErT5). Grey histogram shows isotype control (IgG). f Enrichment score of previously published signatures of cell cycle progression (CCP) [15] and tumour dormancy [16] in untreated LNCaP–iSnail cells (NoDox), and cells treated with dox for 5 days (EMT5) followed by removal for 3, 5, and 20 days (MErT3–20). g Left of dotted line: raw cell number of untreated LNCaP–iSnail cells at 5 and 10 days (No Dox), and cells treated with Dox for 5 and 10 days (EMT-On). Right of dotted line: LNCaP–iSnail cells were treated with Dox for 5 days, re-seeded into new plates, and cell number quantified after an additional 5 and 10 days of Dox treatment (EMT-On) or had Dox removed (MErT-On). One-way ANOVA, *p < 0.0001. Error bars indicate SEM for biological triplicates. h Representative images of LNCaP–iSnail cells treated with Dox for 5 days in a monolayer (top left) prior to seeding as single cells in 3D Matrigel™ assays (top right). LNCaP–iSnail cells were then either maintained in Dox (bottom left) or had Dox removed (bottom right) for an additional 7 days. i Phenogram showing the relative metabolic state of untreated LNCaP–iSnail cells (D0), treated with Dox for 1 and 5 days (E1–E5), and removed for 10 days (M10). x-Axis portrays the basal oxygen consumption rate (OCR) and y-axis the extracellular acidification rate (ECAR)