Figure 2: TGFβ effects on normal human mammary epithelium.

(a) FACS profile of human mammary epithelial cell preparations (depleted of CD45⁺ and CD31⁺ cells) showing the gates used to select normal human mammary epithelial cell subfractions; A: CD49f⁺/EpCAM⁺, B: CD49f⁺/EpCAM^neg/low and C: CD49f⁻/EpCAM⁺, and schematic representation of the in vitro and in vivo experimental approaches that were used to obtain primary and secondary CFCs, respectively. (b) qRT–PCR of the indicated transcripts in epithelial sub fractions as depicted in (a). Expression was normalized against beta-2-microglobulin and displayed as arbitrary units. Error bars represent ±s.d. of three independent experiments. (c) TGFβ (2.5 ng ml⁻¹) was added (at day 0) to flow-sorted subfractions as depicted in (a) and plated at clonogenic densities in the presence of feeder cells (irradiated fibroblasts) in collagen-coated plates for 7–10 days. Colonies were stained with Giemsa and counted. Results are represented as the log₂ fold changes of primary colonies (or CFCs) in TGFβ-treated compared with untreated cells. Error bars represent ±s.d. of three independent experiments. (d) Microscopy images of Giemsa-stained primary colonies from CFC assays of flow-sorted TGFβ- (2.5 ng ml⁻¹) or SB-(10 μM)-treated cells from fractions A or B. Scale bars, 1 mm. (e) Immunostaining for expression of cytokeratin 14 (green) and cytokeratin 18 (red) of primary colonies from CFC assays of flow-sorted TGFβ- (2.5 ng ml⁻¹) or SB (10 μM)-treated fractions A and B cells. Scale bars, 1 mm. (f) Representative images of secondary colonies of TGFβ- (2.5 ng ml⁻¹) or SB (10 μM)-treated mammary epithelial subpopulation A and B. Scale bars, 1 cm. (g) Bar chart showing number and type (according to their morphology) of secondary colonies of a representative secondary CFC assay of TGFβ- and SB-treated mammary epithelial fractions A and B. (h) Immunofluorescence using antibodies against cytokeratin 14 (CK14) and cytokeratin 18 (CK18) of representative colonies derived from (g). Scale bars, 1 mm