Fig. 4

Ectopic expression of GREM1 promotes cancer cell invasion in a zebrafish model. a, b GREM1 overexpression (OE) inhibits BMP-induced SMAD1/5/8 phosphorylation (pSMAD1/5/8 (a)) and the BMP target genes ID1 and ID3 (b) in MDA-MB-231 and M2 cell lines. GAPDH was used as an internal control. The results are expressed as the mean ± s.d., n = 3. Student’s t test, **P ≤ 0.01. c, d GREM1 OE upregulates the expression of EMT transcription factors and markers in M2 (c) and MDA-MB-231 (d) cells. GAPDH was used as an internal control. The results are expressed as the mean ± s.d., n = 3. Student’s t test, **P ≤ 0.01. e, f GREM1 OE induces more clusters formation in M2 cells (e) and promotes the invasion of MDA-MB-231 cells (f) in zebrafish. Left, quantification of the number of extravasated cells/clusters at 5 days post-injection (dpi). Right, representative images: green, vasculature of zebrafish; red, mCherry-labeled cells. The results are expressed as the mean ± s.e.m., n = 2. Student’s t test, **P ≤ 0.01, ***P ≤ 0.001. g Perivitelline space injection of MDA-MB-231 cells supplemented with rhGrem1 (1 μg/ml) increases cell intravasation in zebrafish. Left, representative images: green, vasculature of zebrafish; red, mCherry-labeled cells. Right, quantification of the number of intravasated cells in each embryonic body at 3 days post-injection (dpi). The results are expressed as the mean ± s.e.m., n = 2. Student’s t test, *P < 0.05