Figure 5: Role of TGF-β2 in dormant HEp3 DTCs.

(a) qPCR analysis of TGF β 2 mRNA levels in the indicated cells cultured in serum-free media for 24 h. (b) qPCR analysis of TGF β 2 mRNA levels in BM and lungs mice organs. (For a and b, n = 6 RNA samples per condition were assessed over 3 independent experiments). (c) qPCR for TGF β 2 mRNA in T-HEp3 cells treated with serum-free media (SF) or conditioned media from lung (Lu CM) or BM (BM CM) (24 h). (n = 6 RNA samples were assessed over 3 independent experiments.) (d) Growth after 5 days of T-HEp3 cells on CAMs (2×10⁵ cells per CAM) pre-treated in culture for 24 h and then on the CAM every day with SF, BM CM or Lu CM (n = 11 (SF), 3 (Lu CM), 8 (BM CM) tumour nodules per condition). (e) Immunohistochemistry for P-H3 (upper row) and p27 (lower row) in T-HEp3 tumour nodule sections after treatment with SF (left column), Lu CM (middle column) or BM CM (right column) for 5 days in vivo; scale bar, 40 μm. Arrows: marker+ cells. Lower graphs: quantification of P-H3+ (left) and p27+ (right) cells (n = 100 cells assessed per section. Fifteen sections assessed from three different tumours per condition). (f) Detection of the indicated antigens in lysates of T-HEp3 cells treated with, SF, Lu CM or BM CM immunoprecipitated (IP) with IgG (IP-IgG) or with TGF-β2 antibody (IP-TGF-β2) for 24 h. Top numbers: (ERK/p38) ratio quantification. (g) qPCR for p53 and DEC2 mRNA in T-HEp3 cells treated as in c (n = 6 RNA samples were assessed over 3 independent experiments). (h) Top panel: immunoblot against TGF-β2 in the BM CM after IgG or anti-TGF-β2 immunodepletion. Lower panel: tumour growth of T-HEp3 cells treated with full or TGF-β2-immunodepleted BM CM or with Lu CM supplemented with PBS (CM) or with 2 ng ml⁻¹TGF-β2 (n = 22 (SF), 15 (IP-IgG), 8 (IP-TGF-β2), 7 (Lu CM), 5 (Lu-CM TGF-β2) tumours per condition). (i) Effect of TGF-β1 (2 ng ml⁻¹) on T-HEp3 and dormant D-HEp3 and BM-D1 tumour growth on CAMs for 5 days in vivo (n = 6 (DHEp3-TGF-β1), 9 (DHEp3+TGF-β1), 10 (BM-D1-TGF-β1), 10 (BM-D1+TGF-β1), 13 (THEp3-TGF-β1), 11 tumours per condition). Data in a–c,e and g, represent mean±s.e.m. In a–c,e and g, *P<0.05, **P<0.01 by Mann–Whitney test. In d,h and i *P<0.05, **P<0.01 by One-way ANOVA with Bonferroni’s multiple comparison test. NS, not significant. Uncropped images of blots are shown in Supplementary Fig. 6.