Figure 1: Effects of TGFβ pathway activation on human BTICs.

(a). Schematic representation of the in vitro and in vivo experimental approaches that were used in combination to study TGFβ regulation of BTIC behaviour. (b) MS-IC assay. Bar chart shows log₂ fold changes of M2 mammosphere numbers (which is a read-out of MS-ICs in the M1 cultures) in treated compared with untreated cultures. Error bars represent ±s.d. of at least three independent experiments. **TGFβ-treated versus untreated cultures, P<0.01. *TGFβ-treated versus untreated cultures, P<0.05 (Student's t-test) (c) LDA assay: different doses of single cells from HCC1954 and MDA-MB-231 M1 mammospheres left untreated or treated with TGFβ (2.5 ng ml⁻¹) for 7 days, as indicated, were implanted in the mammary fat pad of NSG mice at limiting dilutions. Bars represent fold change in total number of BTICs in TGFβ-treated M1 as compared with untreated M1 cultures. (d) Same number of single cells from MDA-MB-231- and HCC1945-expressing lenti-HIV-ZsGreen control (vector control), lenti-Tβ1CA (TβCA) or lenti-TβRII-ΔCyt (TβRII-ΔCyt) were grown as M1 mammospheres and left untreated or treated with TGFβ (2.5 ng ml⁻¹) for 7 days as indicated and then processed as in (b). Bar chart shows log₂ fold changes of MS-ICs in M1 mammospheres compared with vector control untreated M1 cultures. Error bars represent ±s.d. of at least three independent experiments. (e) Immunofluorescence analysis for the expression of E-cadherin, N-cadherin and vimentin in HCC1954 cells grown for 7 days as M1 mammospheres in the absence or presence of TGFβ and then for 24 h in attached conditions. Scale bars, 250 μm (left panel). qRT–PCR assays to assess expression of the indicated transcripts in HCC1954 M1 mammospheres treated with TGFβ (2.5 ng ml⁻¹) or of SB (10 μM) for 4 days. Error bars represent ±s.d. of three independent experiments (right panel). (f) FACS plots of CD44-FITC- and CD24-PE-stained HCC1954 and MDA-MB-231 M1 mammospheres left untreated or treated with TGFβ (2.5 ng ml⁻¹) for 7 days. Numbers indicate the percent of CD44⁺/CD24^−/low population.