Fig. 3

Grem1 maintains stemness in M1 cells. a GREM1 overexpression (OE) induces more mammosphere formation in M1 cells. Left, representative images of mammospheres at 7 days. Right, the number of spheres formed per 1000 cells plated. The primary spheres were disintegrated and replated further. Secondary spheres formed were counted. The results are expressed as the mean ± s.d., n = 3. Student’s t test, **P ≤ 0.01. b Pro-mammosphere formation ability of recombinant human Grem1 (rhGrem1) protein (500 ng/ml) can be neutralized by BMP2 (50 ng/ml). Left, representative images of spheres at 7 days; right, the number of spheres formed per 1000 cells plated. The results are expressed as the mean ± s.d., n = 3. Student’s t test, **P ≤ 0.01. c Flow cytometry analysis shows that GREM1 OE in M1 cells increases the stem population (CD44^+/high CD24^−/low). d GREM1 OE in M1 cells upregulates stem cell transcription factors. GAPDH was used as an internal control. The results are expressed as the mean ± s.d., n = 3. Student’s t test, *P < 0.05, ***P ≤ 0.001. e Flow cytometry analysis showing that 2 days of treatment with rhGrem1 (500 ng/ml) or the BMP type I receptor inhibitor LDN193198 (120 nM) also leads to an increase in the CD44^+/high CD24^−/low population