Fig. 3

HDAC8 supports the induction of EMT by suppressing key-MET TFs in BLBC cells surviving cytotoxic therapy. A Assessment of Vim, Wnt5a, Cdh2, Zeb1 mRNA expression levels by qRT-PCR in vehicle- or CAF-treated (48 h) pG-2 cells, with or without Hdac8 silencing. B GSEA profile showing an enrichment of the "HOLLERN_EMT_BREAST_TUMOR_DN" signature (MSigDB: C2 curated gene sets) in pG-2 cells at basal state (veh) compared to CAF-treated. NES: Normalized Enrichment Score. C Aggregate profile showing changes of H3K27ac at TSS regions of strongly downregulated genes in vehicle- and CAF-treated pG-2 cells (normalized counts basemean ≥ 15, p-adj < 0.05, log2FC ≤ -− 1.5). D Differential binding analysis (DiffBind) depicting changes of H3K27ac occupancy between vehicle- and 48 h CAF-treated pG-2 cells. Genomic regions showing significant changes of H3K27ac occupancy at EMT- or MET-associated genes (MSigDB C2, see method section) were labeled with light red and dark red dots, respectively. E Venn diagram showing genes simultaneously downregulated (normalized counts basemean > 15, p-adj < 0.05, log2FC < − 1.5) and loosing H3K27ac at TSS regions (Diffbind parameters: conc > 3, p-adj < 0.05, FC < -0.5) upon CAF treatment in pG-2 cells. F Pathway enrichment analysis showing that genes with loss of expression and H3K27ac enrich for MET- and cell differentiation-associated signatures (labeled in red). Analysis performed with the online tool. G qRT-PCR and ChIP-RT-PCR of epithelial-specific markers (Cldn8, Cldn3) in vehicle- and CAF-treated pG-2 cells. H Venn diagram showing 10 transcription factors (TFs) with simultaneous gene expression (normalized counts basemean > 15, p-adj < 0.05, log2FC < -− 0.7) and H3K27ac occupancy loss (Diffbind parameters: conc > 3, p-adj < 0.05, FC < − 0.5) upon CAF treatment in pG-2 cells. The murine transcription factor list was retrieved from the Animal TFDB3.0 TF database. I qRT-PCR assessing changes of MET-TFs Elf5, Gata3, Rora, Grhl2 upon pG-2 CAF treatment for 48 h, w/ or w/o Hdac8 silencing. Statistical test: A, I one-way ANOVA, G Student's t test. *p-val < 0.05, **p-val < 0.01, ***p-val < 0.005. Error bars are standard error of the mean (SEM)