Isoform switching of FGFRs during TGF-β-induced EMT. (A) The kinetics of FGFR1 and FGFR2 expression were examined in NMuMG cells treated with 1 ng/ml TGF-β for the indicated periods by quantitative RT–PCR analysis. The ratio of the mRNA levels in TGF-β-treated cells compared with non-treated cells is shown. Each value represents the mean±s.d. of duplicate determinations from a representative experiment. Similar results were obtained in at least three independent experiments. (B) Expression of the alternatively spliced forms of FGFR1 and FGFR2 was examined by RT–PCR using RNA samples from NMuMG cells treated with TGF-β for 36 h. A schematic illustration of the primers is shown at the top. The primers for the IIIb and IIIc isoforms were the sp1–ap1 pair and sp2–ap1 pair, respectively. Ig, extracellular immunoglobulin-like domain; KD, kinase domain. FGFR3 and FGFR4 were not detected in NMuMG cells by quantitative RT–PCR. (C) Regulation of FGFR1 expression (left) and FGFR2 expression (right) by TGF-β and/or FGF-2 were examined by quantitative RT–PCR analysis in NMuMG cells. Values were normalized to the housekeeping gene TBP and are indicated as fold differences compared with the non-treated cells. Each value represents the mean±s.d. of duplicate determinations from a representative experiment. Similar results were obtained in three independent experiments. (D) Erk1/2 phosphorylation (p-Erk1/2) by FGF-2 or FGF-7 was examined by immunoblot analysis. NMuMG cells were preincubated with or without TGF-β for 2 days and then stimulated with 30 ng/ml FGF-2 or 30 ng/ml FGF-7 for 15 min. F2, FGF-2; F7, FGF-7.

Activated phenotype of cells generated by treating with FGF-2 and TGF-β. (A) NMuMG cells were incubated for 4 days in the absence or presence of 1 ng/ml TGF-β alone, 30 ng/ml FGF-2 alone, or both TGF-β and FGF-2, and visualized by phase-contrast microscopy. Scale bar indicates 100 μm. (B) Actin cytoskeleton reorganization was visualized by TRITC-phalloidin staining 4 days after TGF-β or TGF-β+FGF-2 treatment. Scale bar indicates 100 μm. (C) NMuMG cells were cultured for 4 days in the absence or presence of TGF-β, FGF-2, or both, and then the monolayers were wounded. After 12 h, the migratory behaviours of the cells were determined and quantified (right). Bars represent the median for each category from a representative experiment. Similar results were obtained in two independent experiments. The median migration distance of the cells was significantly different for non-treated versus TGF-β+FGF-2, TGF-β versus TGF-β+FGF-2, and FGF-2 versus TGF-β+FGF-2 (P<0.001; non-parametric Mann–Whitney U test). Scale bar indicates 100 μm. (D) NMuMG cells were cultured for 4 days in the absence or presence of TGF-β or both TGF-β and FGF-2, and then seeded into cell culture inserts. After 12 h, the cells that had migrated to the opposite side were stained and quantified. Each value represents the mean±s.d. of triplicate determinations from a representative experiment. Similar results were obtained in two independent experiments. (E) NMuMG cells were preincubated with or without the indicated combinations of TGF-β, FGF-2, and 10 μM GM6001 for 5 days, and then mixed in collagen matrices. After polymerization, the mixtures were released from the culture dishes and incubated for an additional 2 days. Three independent experiments were performed in duplicate, and representative results are shown (#1 in blue and #2 in red). (F) The conditioned media from NMuMG cells treated with TGF-β or both TGF-β and FGF-2 were collected and applied to 10% polyacrylamide gel impregnated with 1 mg/ml gelatin. After removing SDS, the gels were incubated overnight and stained with CBB.

Prevention of EMyoT by FGF-2. (A) Immunohistochemical analysis was performed with anti-α-SMA antibody (green) and propidium iodide to detect nuclei (red) in cells treated with 1 ng/ml TGF-β for 3 days and 13 days. (B) Cells were treated with either 1 ng/ml TGF-β or 30 ng/ml FGF-2 alone, or both and then examined for the induction of α-SMA (left) and calponin (right) at the indicated time points by quantitative RT–PCR analysis. Each value represents the mean±s.d. of duplicate determinations from a representative experiment. Similar results were obtained at least three independent experiments. (C) Protein levels of endogenous E-cadherin and α-SMA were analysed by immunoblotting 4 days after treating with combinations of TGF-β, FGF-2, and 10 μM SU5402. α-Tubulin levels were monitored as a loading control for the whole-cell extracts. (D) Phase-contrast micrographs (upper panels) and confocal images after anti-α-SMA antibody staining (green in lower panels) of NMuMG cells. The ligand and inhibitor concentrations were as follows: 1 ng/ml TGF-β, 30 ng/ml FGF-2, 10 μM SU5402, 1 μM PI-103, 1 μM U73122, and 10 μM U0126. Scale bar indicates 100 μm. (E, F) Immunoblot analyses were performed with lysates from NMuMG cells treated with the indicated combinations of ligands and inhibitors. α-Tubulin was used as a loading control (E). The ligands and inhibitors were incubated in culture media containing 10% FBS for 4 days (E), and 2 days (2d in F) or 4 days (4d in F). The same reagent concentrations were used as described in (D). F, FGF-2; Tβ, TGF-β; SU, SU5402; PI, PI-103; U7, U73122; U0, U0126.

Interaction of CtBP1 with δEF1 to prevent α-SMA induction. (A, B) NMuMG cells transfected with either δEF1 siRNA or SIP1 siRNA were stimulated with or without 1 ng/ml TGF-β, 30 ng/ml FGF-2, or both for 24 h and then examined by quantitative RT–PCR analysis for the expression levels of δEF1 (A, left), SIP1 (A, right), and α-SMA (B). NC, control siRNA. Each value represents the mean±s.d. of duplicate determinations from a representative experiment. Similar results were obtained at least three independent experiments. (C) After transfection with different types of siRNAs against δEF1 (δEF1-B and δEF1-C), α-SMA induction by TGF-β was evaluated by immunoblot analysis. α-Tubulin was used as a loading control. The ratio of δEF1 or α-SMA to α-tubulin was validated by densitometric analysis and shown at the lower panels. NC, control siRNA; Tβ, TGF-β; F2, FGF-2. (D) NMuMG cells transfected with control siRNA (NC) or two different types of CtBP1 siRNAs (#1 and #2) were stimulated with TGF-β, FGF-2, or both for 60 h as indicated. After measuring the protein concentrations, the cell lysates were examined by immunoblot analysis. α-Tubulin was used as a loading control. (E) After NMuMG cells were incubated in culture media containing 10% FBS for 4 days with the indicated ligands, cell lysates from ∼1 × 10⁶ cells were examined for protein concentrations and then subjected to immunoprecipitation with normal rabbit IgG (NRS) or an anti-δEF1 antibody. Copurified CtBP1 was detected by immunoblotting with an anti-CtBP1 antibody (top panel). The expression levels of δEF1, CtBP1, and α-tubulin in the same lysates were verified by immunoblotting (lower three panels). The ratio of δEF1 to α-tubulin was validated by densitometric analysis and shown at the bottom.

Effects of TGF-β and FGF signalling on cancer cell invasion. (A) NMuMG cells were pretreated with 1 ng/ml TGF-β (NMu-β) or both 1 ng/ml TGF-β and 30 ng/ml FGF-2 (NMu-fβ) for 2 days and then seeded on collagen gels. After 1 week, the gels were fixed, embedded in paraffin, and stained with HE. Bars represent the median for each category from a representative experiment. Similar results were obtained in two independent experiments. Scale bar indicates 100 μm. (B) MCF-7-GFP cells were seeded on a collagen gel and treated with 1 ng/ml TGF-β or 30 ng/ml FGF-2+TGF-β (i–vi). NMuMG cells were pretreated with 1 ng/ml TGF-β (NMu-β cells) or 30 ng/ml FGF-2 together with TGF-β (NMu-fβ cells) for 2 days, and mixed with MCF-7-GFP cells in the presence or absence of TGF-β and TGF-β+FGF-2 (vii–xii). After 7 days, the gel was fixed and stained with HE (i, iii, v, vii, ix, xi). GFP was detected by confocal laser scanning microscopy (ii, iv, vi, viii, x, xii). Scale bars indicate 100 μm. (xiii, xiv) Statistical analyses were performed using a non-parametric Mann–Whitney U test. The median migration distance of cells was significantly different at P>0.05 between the two groups of MCF-7 cells (non-treatment versus TGF-β+FGF-2 treatment, and TGF-β treatment versus TGF-β+FGF-2 treatment) and at P<0.001 between the two groups of MCF-7 cells mixed with the indicated cells (NMuMG cells versus NMu-fβ cells and NMu-β cells versus NMu-fβ cells). Bars represent the median for each category from a representative experiment. (C) NMuMG cells were cultured for 4 days in the absence or presence of TGF-β or both TGF-β and FGF-2, and then seeded into collagen gel-loaded culture inserts with MCF-7-GFP cells, which had been cocultured in cell culture inserts with NMuMG cells. The GFP-positive cells that had invaded to the opposite side were fixed and photographed (left), and typical morphology of the cells were counted and quantified (right).

Schematic illustration of EMT induction by TGF-β and FGF-2. ‘Epithelial cells' differentiate into ‘fibroblastic cells' through EMT induced by TGF-β, and further differentiate into α-SMA-positive ‘myofibroblastic cells' through EMyoT. When FGF-2 is present in this process, FGF-2 induces the differentiation of (myo)fibroblastic cells to ‘activated fibroblastic cells'. The red dot in CtBP1 indicates phosphorylation.