Mdm2 triggers the ubiquitination of NUB1.

<p>(A) HEK-293T cells were transfected with Myc-NUB1 alone or together with Mdm2, and twenty-four hours later lysates were prepared using an hypotonic / Triton X-100 buffer supplemented or not with N-Ethylmaleimide (NEM). Proteins were separated through SDS-PAGE and NUB1 was detected by western blotting using the anti-Myc antibody. (B) HEK-293T cells were transfected with Myc-NUB1 alone or together with wild type (WT) or catalytically inactive (C462A) Mdm2. Lysates were prepared and proteins were separated through SDS-PAGE and analyzed by western blotting with the appropriate antibodies.</p

NUB1 is ubiquitinated by Mdm2 on lysine 159.

<p>(A) Workflow of the protocol used to identify NUB1 ubiquitinated lysine. (B) METTRE SPECTRES (C) HEK-293T cells were transfected with wild type (WT), K134R, K159R or K134,159R (2KR) mutants Myc-NUB1 together with 6HF-Ubiquitin and Mdm2 constructs. 6HF-Ubiquitinated proteins were isolated by Ni2+ pull down and NUB1 was revealed by western blotting using an anti-NUB1 antibody. (D) Lysates from HEK-293T cells expressing Mdm2 alone or together with wild type (WT), K134R or K159R mutants Myc-NUB1 were subjected to immunoprecipitation with the anti-Myc 9E10 antibody. Immunoprecipitates and input samples were analyzed by western blotting using an anti-Mdm2 antibody.</p

Mdm2 interacts with NUB1.

<p>(A) Lysates from HEK-293T cells expressing Myc-NUB1 alone or together with Mdm2 were subjected to immunoprecipitation with an anti-Mdm2 antibody. Proteins were separated through SDS-PAGE and NUB1 was detected by western blotting using an anti-Myc antibody (9E10). Amount of precipitated Mdm2 and expression levels of both proteins in cell extracts were controlled. (B) Lysates from HEK-293T cells expressing Mdm2 alone or together with Myc-NUB1 were subjected to immunoprecipitation with the anti-Myc 9E10 antibody. Immunoprecipitates and cell extracts were analyzed by western blotting using an anti-Mdm2 and anti-Myc antibodies. * Shifted NUB1.</p

Mdm2-mediated ubiquitination controls NUB1 activity and is not a proteolytic signal.

<p>(A) HEK-293T cells were transfected with Myc-NUB1 and 6HF-Nedd8, alone or together. Twenty-four hours post-transfection, cells were treated with 30 μM of MG132 for 4 hours where indicated or with DMSO (vehicle) as a control. 6HF-neddylated proteins were revealed by western blotting using the anti-Flag M2 antibody. (B) HEK-293T cells were transfected with 6HF-Nedd8 and Mdm2, alone or together with WT or K159R Myc-NUB1. Twenty-four hours post-transfection, proteins were separated through SDS-PAGE and 6HF-neddylated proteins were revealed by western blotting using the anti-Flag M2 antibody. (C) HEK-293T cells were transfected with GFP-HHT97 in combination with Myc-NUB1 WT or K159R. 24 hours after transfection cells were dispatched in 6-wells plates and allowed to adhere for 12 more hours. Cells were then treated with 20 μg/ml of Cycloheximide and harvested at indicated time points. Amount of remaining HTT97 was evaluated by western blot of cell lysates (left panel). Densitometry analysis of three independent experiments have been performed and used to establish the half-life of HTT97 and to evaluate the impact of NUB1 WT or K159R expression (right panel).</p

Molecular characterization of Mdm2-dependant ubiquitination of NUB1.

<p>(A) HEK-293T cells were transfected as indicated with Myc-NUB1, Mdm2 together with wild type (WT) or lysine-less mutant (K0) 6HF-Ubiquitin expressing constructs. Twenty-four hours post-transfection, 80% of cells were lysed in Guanidine-HCl containing buffer and 6HF-Ubiquitinated conjugates were isolated on Ni2+-NTA agarose beads. The remaining 20% were lysed in non-denaturing buffer to detect non-modified NUB1. Purified 6HF-Ubiquitinated proteins and cell extracts were resolved by SDS-PAGE and NUB1 was revealed by western blotting using an anti-NUB1 antibody. (B) HEK-293T cells were transfected with Myc-NUB1 and Mdm2, together with wild type (WT), K48R or K11R mutants 6HF-Ubiquitin. Ubiquitination of NUB1 was analyzed as in (A). (C) HEK-293T cells were transfected in 10-cm dishes with Myc-NUB1 alone or together with Mdm2. Twenty-four hours post-transfection, cells were split into six-well plates and allowed to rest overnight. Cells were then treated by supplementation with 100 μg/ml of cycloheximide (CHX) in their culture medium for the indicated time lapses. NUB1 stability was monitored by western blotting using the anti-Myc 9E10 antibody.</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

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

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

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

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