Fig. 5

GREM1 knockdown in 19TT breast cancer-associated fibroblasts (CAFs) attenuates fibrotic characteristics. a qRT-PCR comparison of relative GREM1 expression in M2, MCF7, MDA-MB-231, HM, W18, and W21 fetal mesenchymal stem cells (MSCs), foreskin fibroblasts, and 19TT CAFs. 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, ***P ≤ 0.001. b qRT-PCR analysis of the selected genes, BMP targets, TGFβ pathway constituents/targets, fibroblast activation markers, and matrix metalloproteinases in 19TT CAFs with/without shRNA-mediated GREM1 knockdown. 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 ≤ 0.01, ***P ≤ 0.001. c Western blot analysis to detect the changes in indicated proteins after GREM1 knockdown in 19TT CAFs. d 19TT CAFs with/without GREM1 knockdown were stained with fluorescein-phalloidin (green) to visualize F-actin. DAPI was used for nuclear staining (blue). e Collagen gel contraction assay. 19TT CAFs with/without GREM1 knockdown were embedded in collagen gels. After 24, 48, and 72 h, the area of each gel (white dash circle) was imaged and quantified. Left, representative images of contracted gels. Right, the percentage of gel contraction. Quantification is shown in the “Methods” section. The results are expressed as the mean  ± s.d., n = 3. Student’s t test, **P ≤ 0.01