Förster S , Koziol U , Schäfer T , Duvoisin R , Cailliau K , Vanderstraete M , Dissous C , Brehm K .

107475/Z/15/Z Wellcome Trust , Wellcome Trust

	Fig 1. Domain structure of E. multilocularis FGF receptors.Schematic representation of the domain structure of the E. multilocularis receptors EmFR1, EmFR2, and EmFR3 according to SMART (Simple Modular Architecture Tool) analyses. As a comparison, the structure of the human FGFR1 (HsFGFR1) is shown. Displayed are the location and size of the following predicted domains: TK domain (TKD) in purple and IG-domain (Ig) in orange. Predicted signal peptides are depicted in red and transmembrane domains are shown as blue bars. The numbers of amino acids in full length receptors are shown to the right.
	Fig 2. WMISH analysis of emfr1 expression.Left panel, antisense probe detecting emfr1 expression. Right panel, sense probe (negative control). WMISH signal is shown in green, nuclear DAPI staining in blue. gl, germinal layer; ps, developing protoscolex. Bar: 20 μm.
	Fig 3. WMISH analysis of emfr2 expression.Shown are WMISH analyses of emfr2 expression (A, B) and co-detection of emfr2 WMISH and EdU incorporation (C, D, E) during metacestode development. In C, D, and E, metacestodes were cultured in vitro with 50 μM EdU for 5 h and were then processed for WMISH and EdU detection. In all panels the WMISH signal is shown in green, EdU detection in red, and DAPI nuclear staining in blue. A, detail of the germinal layer. B, general view of a region of a metacestode with protoscoleces in different stages of development. C, early protoscolex development. D, late protoscolex development. E, Detail of a sucker, maximum intensity projection of a confocal Z-stack. r, rostellum; s, sucker. Bars are 20 μm for A, 50 μm for B, 10 μm for C, and 50 μm for D. Sense probe (negative control) was negative in all samples.
	Fig 4. Co-detection of emfr3 WMISH and EdU incorporation during metacestode development.Metacestodes were cultured in vitro with 50 μM EdU for 5 h and were then processed for WMISH and EdU detection. In panels displaying multiple channels, the WMISH signal is shown in green, EdU detection in red, and nuclear DAPI staining in blue. A, germinal layer. B, developing protoscolex. Arrowheads indicate Edu+ emfr3+ double-positive cells. Bars are 25 μm for A, 50 μm for B (main panel) and 10 μm for B (enlarged inset).
	Fig 5. Effects of host FGFs on E. multilocularis proliferation and development.A, effects of FGFs on metacestode proliferation. Axenically cultivated metacestode vesicles (eight per well) were incubated for 48 h in DMEM medium (without FCS), then BrdU (1 mM) and FGFs (10 or 100 nM) were added and incubation proceeded for another 48 h before cell lysis, DNA isolation, and BrdU detection. Bars represent percentage of BrdU incorporation with the cMEM control set to 100%. Statistical evaluation of four independent experiments (n = 4) which were conducted in duplicates is shown. Student’s t-test (two tailed): *p<0.05. B, effects of host FGFs on metacestode vesicle development. Single axenically generated metacestode vesicles were in vitro cultivated for four weeks in the presence of 10 nM FGF1 (aFGF) or 5 nM FGF2 (bFGF) with daily medium changes. Control vesicles were kept in cMEM. Growth (in ml) of vesicles was monitored. In each of three independent experiments (n = 3), four vesicles were examined for every single condition. C, effects of FGFs on E. multilocularis primary cell proliferation. Freshly isolated E. multilocularis primary cells were incubated for 48 h in 2 ml cMEM, then BrdU (1 mM) and FGFs (100 nM) were added and cells were incubated for another 48 h before DNA isolation and BrdU detection. Statistical analysis was performed as in A. D, effects of host FGFs on metacestode vesicle development. Freshly prepared E. multilocularis primary cells were cultured for 21 days in cMEM medium in the presence or absence of 100 nM FGF1 (aFGF) or FGF2 (bFGF). Half of the medium volume was renewed every second day. The number of newly formed metacestode vesicles at day 21 was analysed. The bars represent the percentage of formed vesicles with the cMEM control set to 100%. The statistical evaluation of three independent experiments (n = 3) which were conducted in duplicates is shown. Student’s t-test (two-tailed): *p<0.05.
	Fig 6. Effects of BIBF 1120 on E. multilcoularis metacestode vesicles and primary cells.A, axenically cultivated metacestode vesicles (8 per well in 2 ml volume) were incubated for 18 days in the presence of 0.1% DMSO or BIBF 1120 in different concentrations (as indicated) and the structural integrity of the vesicles was monitored. Structurally intact vesicles (B) and damaged vesicles (C) are shown to the right. The experiment was repeated three times in duplicates. D, freshly isolated E. multilocularis primary cells were cultured for 21 days in cMEM medium in the presence of DMSO (0.1%) or BIBF 1120 at different concentrations (as indicated). After 21 days, newly formed metacestode vesicles were counted. The DMSO control for each of the three independent experiments was set to 100%. Microscopic images (25x magnification) of cultures after 2 weeks with DMSO (E) or 10 μM BIBF 1120 (F) are shown to the right.
	Fig 7. Effects of host FGFs and BIBF 1120 on EmMPK1 phosphorylation in metacestode vesicles.A, axenically cultivated metacestode vesicles were incubated in cMEM (control) or in medium without FCS (0 s), upon which FGF1 (aFGF) or FGF2 (bFGF) were added at a concentration of 10 nM for 30 sec (30s), 60 sec (60s) or 60 min (60min). Protein lysates were subsequently separated by 12% SDS-PAGE and Western blots were analysed using polyclonal antibodies against Erk-like MAP kinases (anti-ERK) or double phosphorylated Erk-like MAP kinases (anti-p-ERK). B, axenically cultivated metacestode vesicles were incubated with DMSO (negative control), 5 mM or 10 mM BIBF1120 (30 min each) and cell lysates were subsequently analysed as described above. Both experiments were performed in triplicate with similar results.

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