Figure 4: Relief of TGFβ-mediated negative regulation of growth releases ER^pos cells from quiescence.

(a) Phase contrast micrographs (left column) after 8 days in culture and immunoperoxidase staining with Ks20.8 (middle column) and ER (SP1 prediluted, right column) of primary cultures of CD166^high-derived cells in FAD2 (upper panel) or TGFβR2i (lower panel). Nuclei are counterstained with haematoxylin. Whereas Ks20.8^pos cells remain quiescent and ER negative on FAD2, they are colony forming and ER positive in TGFβR2i. Scale bar, 50 μm. (b) Quantification at day 13 of ER^pos (closed bar) or Ks20.8^pos (open bar) colony-forming units (CFUs) derived from CD166^high/CD117^low cells from three consecutive biopsies (p957, p958 and p959) in FAD2 or TGFβR2i. In all three cases TGFβR2i supplied throughout 9–11 days supported colony formation of Ks20.8^pos/ER^pos cells. (c) qRT-PCR of ER (ESR1), K8 (KRT8), FOXA1, ELF5 and K18 (KRT18) of RNA extracted from second-passage cells cultured for 5 days in FAD2 (open bars), in FAD2 with SB421543 (shaded bars) and in TGFβR2i (solid bars), respectively. Note the collective upregulation of ER and ER-associated gene expression in TGFβR2i. Error bars indicate s.d. of three technical triplicates. (d) Western blotting of proteins extracted at day 6 from second-passage CD166^high/CD117^lowcells seeded at 4,000 cells per cm² all cultured without EGF in FAD2 (left lane), in FAD2 with SB421543 (middle lane) and in TGFβR2i (right lane), respectively, incubated with antibodies recognizing phosphorylated SMAD2 (upper panel), SMAD2/3 (second panel), ER (third panel) and β-actin (lower panel). TGFβR2i inhibits pSMAD2 and upregulates ER protein expression.