Enhanced Met expression levels in <i>Del-R26</i>^<i>Met</i> myoblasts does not perturb activation of downstream signalling effectors.

<p>Limb transverse sections of E10.5 control and <i>Del-R26</i>^<i>Met</i> embryos showing the distribution of phospho-Met (on Tyr_1234–1235), phospho-Akt, phospho-ERKs (red) and of Pax3 protein (green) in myoblasts. Note ectopic phospho-Met in limb mesenchyme (arrowheads) and in non-migrating myoblasts (arrows) in <i>Del-R26</i>^<i>Met</i> mutants. Asterisks indicate non-specific staining in blood cells. Scale: 100μm.</p

Ubiquitously excess wild-type Met in developing embryos results into hyperflexed forelimbs.

<p>(A) Top: <i>Del-R26</i>^<i>Met</i> and control P0 mice showing hyperflexed limbs in mutants. Bottom: anti-myosin heavy chain II IHC using MF20 antibodies on forelimb transversal sections of P0 <i>Del-R26</i>^<i>Met</i> and control mice at the level of the forearm showing almost absence of extensor (asterisk) and a great reduction of flexor (arrowhead) muscle mass in mutants. (B, C) Whole mount ISH with <i>MyoD</i> probe of E12.5 embryos (B) and β-galactosidase staining of E11.5 embryos (C) showing that developing appendicular muscles are reduced in <i>Del-R26</i>^<i>Met</i> embryos (limbs are outlined in panels). The arrowhead in bottom panel B indicates developing ventral limb muscles (flexor). Scale: 500μm.</p

Schematic representation summarizing the different molecular and phenotypic effects of enhanced Met expression in myoblasts and limb mesenchymal cells.

<p>In a wild-type context (top), limb mesenchymal cells secrete HGF required for migration of myoblasts towards the limb buds. Enhanced expression of Met in myoblasts (as assessed in <i>Pax3-R26</i>^<i>Met</i> embryos; middle) does not alter their migration due to a buffering event: the activation levels of signalling effectors such as ERKs and Akt are restrained despite enhanced Met phosphorylation. The size of each signal is representative of their phosphorylation levels. Limb mesenchymal cells are vulnerable to ectopic Met expression (as assessed in <i>Prx1-R26</i>^<i>Met</i> embryos; bottom), illustrated by changes in gene expression, by failure of HGF bioavailability, and by myoblast migration defects. Alteration of HGF bioavailability can be due to: 1) upregulation of a negative interactor that would interfere with the capacity of HGF to bind/activate Met (indicated as “HGF inhibitor”), 2) downregulation of a HGF interactor acting as enhancer of its bioactivity (indicated as “HGF activator”), 3) expression of a chemorepellent factor that renders limb mesenchyme inaccessible to migrating myoblasts, or 4) HGF titration by ectopic Met in mesenchymal cells (indicated as “HGF trapping”).</p

Ectopic Met in limb mesenchyme alters HGF bioavailability.

<p>(A) Scheme illustrating the experimental procedure employed for evaluating through MDCK cell scattering the bioavailability of HGF from control and <i>Del-R26</i>^<i>Met</i> mutant limb mesenchymal cells or from dissected forelimbs. The scheme indicates the experimental procedure applied for collecting media conditioned by limb mesenchymal cells for biochemical analysis (top; shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005533#pgen.1005533.s009" target="_blank">S9A Fig</a>), for MDCK scattering assays using co-cultures with limb mesenchymal cells (middle; shown in Fig 9C, 9D and 9E) or with dissected limbs (bottom; shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005533#pgen.1005533.s009" target="_blank">S9B Fig</a>). (B) Pictures of MDCK colonies showing the three categories that were defined to determine the extent of cell contact and spreading for quantification studies of scattering response. (C) Quantitative analysis of MDCK cell scattering in co-cultures with control limb mesenchymal cells in the absence (no) and in the presence of the Met inhibitor PHA665752 (PHA; 1μM), cryzotinib (Cryzo; 1μM), or SU11274 (SU; 1μM). (D) Quantitative analysis of MDCK cell scattering in co-cultures with control limb mesenchymal cells in the absence (no) and in the presence of the anti-HGF blocking antibodies (anti-HGF; 30μg/ml). (E) Quantitative analysis of MDCK cell scattering in co-cultures with control or <i>Del-R26</i>^<i>Met</i> limb mesenchymal cells. Note a drastic reduction in the scattering response when MDCK cells are co-cultured with <i>Del-R26</i>^<i>Met</i> mutant cells (control: n = 4; <i>Del-R26</i>^<i>Met</i>: n = 3). Mann-Whitney and Student-<i>t</i> test.</p

Myoblast migration is impaired in <i>Del-R26</i>^<i>Met</i> mutants.

<p>(A, B) Whole mount ISH of E10.5 embryos with <i>Lbx1</i> (A) and <i>Pax3</i> (B) probes showing drastic reduction of migrating myoblasts towards the developing tongue (arrowhead), fore and hind limbs. Bottom panel reports an enlargement at forelimb levels. (C, D) Quantification analyses of <i>Lbx1</i> (C) and <i>Pax3</i> (D) positive domains in forelimbs. Left panels: each plot represents the average signal distribution along the white line in forelimbs. Right panels: quantifications and statistical analyses of the sum of signal intensity based on intensity plots in left panels. Numbers of samples for <i>Lbx1</i>: control, n = 13; <i>Del-R26</i>^<i>Met</i>, n = 4; for <i>Pax3</i>: control, n = 11; <i>Del-R26</i>^<i>Met</i>, n = 8. The sum of <i>Pax3</i> signal intensity was calculated between point A and B: A indicating a fixed position between the somites and the limb whereas B being placed at a fixed distance from A. Note almost lack of signal in <i>Del-R26</i>^<i>Met</i> mutants. Scale: 500μm. Mann-Whitney and Student-<i>t</i> test.</p

Ectopic Met in limb mesenchyme down-regulates the expression levels of <i>Notum</i> and <i>Syndecan4</i>.

<p>qRT-PCR analysis of transcript levels of mouse <i>Met</i> (<i>mMet</i>), <i>Pax3</i>, <i>Notum</i>, and <i>Syndecan4</i> (<i>Sdc4</i>) in controls (ctrl; n = 11), <i>Del-R26</i>^<i>Met</i> (Del-Met; n = 11), <i>Met</i>^{<i>LacZ/d(neo)</i>} (KO; n = 7). Each dots corresponds to transcript levels in forelimbs of E10.5 individual embryos (done in triplicate). Columns correspond to the average value, expressed as mean ± s.e.m. Note: downregulation of <i>mMet</i> and <i>Pax3</i> in <i>Del-R26</i>^<i>Met</i> and <i>Met</i>^{<i>LacZ/d(neo)</i>} mutants compared to control, consistent with lack of migrating myoblasts; downregulation of <i>Notum</i> and <i>Syndecan4</i> in <i>Del-R26</i>^<i>Met</i> mutants compared to control, whereas no significant changes were found in <i>Met</i>^{<i>LacZ/d(neo)</i>} mutants. Mann-Whitney and Student-<i>t</i> test.</p

Triflorcas alters the phosphorylation status of cell cycle-related proteins.

<p>(A) The phosphorylation status of several cell cycle proteins was analyzed by applying the phospho-array KPSS 10.1 (Kinexus Bioinformatics). GTL-16 cells were treated with vehicle (control) or Triflorcas (TFC; 3 µM) for 72 hours (left and right panels, respectively). Red circles highlight constituents of the Akt/mTOR/S6 pathway. Green and blue circles surround nucleophosmin/B23 and Rb phospho-epitopes, respectively. (B) The graph shows the ratio of phosphorylation levels of the indicated proteins in cells untreated versus those exposed to Triflorcas. (C) Phosphorylation levels of nucleophosmin/B23 at Ser₄ and Rb at S₇₈₀ were increased and decreased, respectively, in GTL-16 cells exposed to Triflorcas (TFC; 3 µM for 24 hours).</p

Triflorcas perturbs cell cycle progression, leading to mitotic failure.

<p>(A) Graph showing the ratio of percentage of GTL-16 cells in G0/G1 phase over the percentage of cells in S+G2/M phase. Cells were treated with Triflorcas (TFC; 3 µM), or vehicle (cntr) for 48 hours, then stained with propidium iodide. Values are expressed as means ± s.e.m. ***P<0.001; Student-<i>t</i> test (n = 3). Percentages of cells in G0/G1, S, and G2/M phases are reported in the table. (B) GTL-16 cells were grown in the presence of Triflorcas or vehicle and nuclei were visualized using DAPI staining. Arrows show example of cells with multiple nuclei (40X magnification). (C) Quantification of cells with mitotic failure expressed as percentage of the total number of cells analyzed. Triflorcas (TFC; 3 or 10 µM), but not SU11274 (SU; 1 µM), crizotinib (crizo; 1 µM), or PHA665752 (PHA; 1 µM) led to a significant increase in the number of cells with mitotic failure. Values are expressed as means ± s.e.m. ***P<0.001; Student-<i>t</i> test.</p

Triflorcas elicits a selective gene expression profile on stress and toxicity pathways.

<p>(A) The expression profile of 84 genes related to cell stress and toxicity was analyzed in GTL-16 cells. Cells were treated with either Triflorcas (black columns; 3 µM) or SU11274 (grey columns; 1 µM) for 24 hours, and gene expression was compared to that of untreated cells. Genes were grouped in clusters corresponding to oxidative/metabolic stress, heat shock, proliferation/carcinogenesis, growth arrest/senescence, inflammation, and apoptosis/necrosis signaling. Only statistically significant changes in gene expression are indicated (<i>P</i><0.05). Triflorcas altered the expression of only 14 genes compared to the alteration of 39 genes induced by SU11274 treatment. Notably, the expression of <i>cyp1A1</i> was increased 611-fold in the presence of Triflorcas. (B) Western blot analysis showing the up-regulation of CYP1A1 protein levels in cells exposed to Triflorcas (3 or 10 µM). Acacetin (ACA; 10 µM) treatment prevented CYP1A1 up-regulation by Triflorcas (TFC). (C) CYP1A1 up-regulation by Triflorcas also occurred in ErbB1-addicted cancer BT474 cells. Gefitinib (Gef; 10 µM) and SU11274 (SU; 2 µM) were used as controls.</p

Triflorcas blocks Met-triggered cell survival and in vitro tumorigenesis of human NSCLC cells carrying Met mutations (H2212 and H1437) and of human gastric carcinoma cells carrying Met amplification (GTL-16).

<p>(A) Survival of H2122 and GTL-16 cells was reduced by Triflorcas at indicated concentration (µM; n = 3). In contrast, SU11274 (SU), crizotinib (crizo), and PHA665752 (PHA) impaired survival of GTL-16, but not of H2212 cells. Cells were serum-starved for 24 hours and then incubated with compounds for 48 hours. (B) Triflorcas blocked anchorage-independent growth of H1437 and GTL-16 cells in a dose dependent manner (n = 3). SU11274, crizotinib, and PHA665752 impaired in vitro tumorigenesis of GTL-16, but not of H1437 cells. Values are expressed as means ± s.e.m. **P<0.01; ***P<0.001; Student-<i>t</i> test.</p