Fig. 2

TGFβ secreted by breast cancer cells and inflammatory cytokines induce GREM1 expression in CAFs. a GREM1 expression in 19TT CAFs after treatment with conditioned medium (CM) from breast cell lines (M1, MDA-MB-21, or MCF7). The expression was normalized to the parallel time control of normal medium treatment. The results are expressed as the mean  ± s.d, n = 3. Student’s t test, *P < 0.05, ***P ≤ 0.001. b TGFB1, TGFB2, TGFB3, TNFA, and IL1B mRNA levels in 52 breast cancer cell lines. The expression levels were categorized into four groups: background, low, intermediated, and high. c TGFB1/2/3, TNFA, and IL1B expression in primary breast cancer samples. The expression level was categorized into four groups: background, low, intermediated, and high. d TGFβ3 (5 ng/ml), TNFα (10 ng/ml), or IL1β (10 ng/ml) induce GREM1 expression in 19TT cancer-associated fibroblasts (CAFs). Expression was normalized to the parallel time control of buffer treatment. The results are expressed as the mean  ± s.d., n = 3. Student’s t test, *P < 0.05, **P ≤ 0.01, ***P ≤ 0.001. e Measurements of TGFβ activity in CM from breast cancer cell lines using a CAGA luciferase (LUC) reporter assay in HEK293T cells as a readout. TGFβ-neutralizing antibody (10 ng/ml) was added to demonstrate that luciferase activity in CM is due to the TGFβ activation and not activins or nodal. Recombinant TGFβ was added to control for the functionality of the assay. The value is normalized to β-galactosidase (βGal) activity. The results are expressed as the mean ± s.d, n = 3. Student’s t test, ***P ≤ 0.001. f The induction of GREM1 expression in 19TT CAFs by CM from MCF7 and MDA-MB-21 is blocked by TGFβ-neutralizing antibody. The results are expressed as the mean ± s.d, n = 3. Student’s t test, **P ≤ 0.01