Supplementary Figure 4: TGFβ1 & 2 expressions and signalling in HEp3-derived cell lines.

(a) Affymetrix array values for the expression of TGFβ2 mRNA in D-HEp3, T-HEp3 and R-HEp3 (D-HEp3 cells that resumed growth after a prolonged dormancy ∼ 2 months). (b) D-HEp3 cells were treated with a TGFβ receptor I inhibitor (LY-364947, 5μM) for 2, 4 and 6 h in serum free conditions and whole cell lysates were immunoblotted for P-p38 and p38α. (c) qPCR analysis of DEC2 mRNA expression in D-HEp3 cells treated with the TGFβRI inhibitor (LY-364947, 5μM) for 24 h in serum free conditions. n = 6 RNA samples per condition were assessed over 3 independent experiments, error bars denote s.e.m., *p<0.05 by Mann Whitney test. (d) D-HEp3 cells were treated with the TGFβRI inhibitor (LY-364947, 5 μM) for 24 h and then inoculated on CAM (2×10⁵ cells/animal) and then treated with the LY-364947, 5 μM on the CAM every 24 h. 4 days later tumours were minced and the number D-HEp3 cells quantified in collagenase suspensions. Graph: number of cells/tumour, n = 6 tumour nodule assessed per condition, p = 0.0011 by Mann Whitney test. (e) qPCR for DEC2 in 4T1 cells treated with BM CM for 24 h. n = 6 RNA samples per condition were assessed over 3 independent experiments. Error bars denote s.e.m., *p<0.05 by Mann Whitney test (f) Representative cleaved caspase-3 (C-C3) staining in T-HEp3 tumours after treatment with either serum free, lung CM or BM CM for 4 days in vivo. Scale bar: 40 μm. Lower right graph: quantification of Cleaved Caspase-3 (C-C3) positive cells. Y axis: number of C-C3 positive cells per field of view (FOV), mean±s.e.m. (n = 100 cells assessed/section. 15 sections assessed from 3 different tumour/condition). (g) Top panel: IB against TGFβ1 after control IgG or anti-TGFβ1 IgG immunodepletion of the BM CM. Lower panel: Tumour growth of T-HEp3 cells treated with full (IP-IgG) or TGFβ1 immunodepleted (IP-TGFβ1) BM CM for 5 days in vivo, n = 6 (SF), 8 (IP-IgG), 9 (IP-TGFb1) tumour nodules assessed, *p<0.05 by One-way ANOVA-Bonferroni’s multiple comparison test. Right panel: IB showing p38α activation after treatment of T-HEp3 cells with full (IP-IgG) or TGFβ1 depleted (IP-TGFβ1) BM CM for 24 h.