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. 2020 Apr 21;12(4):1019. doi: 10.3390/cancers12041019

Figure 7.

Figure 7

BMP pathway activity partially sustains a mesenchymal state of pancreatic cancer cells and BMP signature gene expression occurs in developmental instances of EMT. (a) Western blot analyses of proteins indicated on the right. PANC-1 cells were treated with DMSO or 50 nM LDN193189 (LDN) for 72 h as indicated. Positions of molecular weight (MW) standards in kDa are given on the left. E-CADHERIN could not be detected. Detection of ACTIN and GSK3β was used as control for equal loading. Proteins were detected using whole-cell lysates, except for FOXA1, ID1, ID2, ZEB1 and GSK3β for which nuclear extracts were used. (b) qRT-PCR analyses of mRNA expression in PANC-1 cells treated with DMSO or 50 nM LDN193189 (LDN) for 72 h as indicated. Significance was determined using two-tailed one sample t-test. Shown is the mean+SEM; n = 4. Rel. expr.: relative expression normalized to that of GAPDH. ns: not significant. *: p < 0.05, **: p < 0.01. (c) Phase contrast images of PANC-1 cells treated with DMSO or 50 nM LDN193189 (LDN) for 72 h as indicated. Two representative fields of view are shown for each condition. Scale bar: 100 µm. (d) Gene set enrichment analysis (GSEA) of BMP signature genes in publicly available sets of genes regulated in EMT-associated processes in different organisms in vivo. Only genes upregulated by Snail1-HA in LS174T cells were considered. Source publications of the analyzed gene sets are given on the left. Plotted is the negative log10 of the adjusted (adj.) p-value. Vertical dotted line indicates the significance threshold of adjusted (adj.) p-value ≤ 0.05. A detailed list of GSEA results is given in Table S4. (e) Model depicting the proposed role of BMP signaling in EMT execution downstream of SNAIL1. Upon induction of EMT and upregulation of SNAIL1, BMP pathway-associated SMAD transcription factor complexes are activated and control a subset of critically EMT-relevant genes. Examples for such BMP signature genes are shown. Note that SNAIL1 regulates additional epithelial and mesenchymal marker genes independently of BMP pathway activity.