Journal: PLoS Genetics

Article Title: Tissue-Specific Gain of RTK Signalling Uncovers Selective Cell Vulnerability during Embryogenesis

doi: 10.1371/journal.pgen.1005533

Figure Lengend Snippet: (A) Western blot analyses of total protein extracts of E15.5 primary embryonic hepatocytes derived from R26 stopMet (containing the LacZ-stop cassette therefore expressing the β-galactosidase) and from Del-R26 Met (after deletion of the LacZ-stop cassette, therefore expressing the transgenic Met detected by anti-human Met antibodies) embryos. Analyses was performed before and after HGF stimulation (50ng/ml). Note: a) similar levels of mouse Met in R26 stopMet and Del-R26 Met cells; b) high levels of Met phosphorylation on Tyr 1234–1235 in Del-R26 Met cells independently of HGF stimulation; c) comparable Gab1, Akt, Stat3, and ERK phosphorylation levels upon HGF stimulation in R26 stopMet and Del-R26 Met cells; d) despite Gab1 phosphorylation in untreated Del-R26 Met cells in contrast to control R26 stopMet cells, the phosphorylation levels of Akt, Stat3, and ERK is unchanged. Actin protein levels were used as loading controls in all western blot analyses. (B) Left: western blot analyses of total protein extracts of E15.5 primary embryonic hepatocytes derived from Del-R26 Met embryos. Note basal levels of Met phosphorylation on Tyr 1003 and on Tyr 1349 ; phosphorylation levels are further increase upon HGF stimulation (50ng/ml). Right: schematic representation of Met indicating the different tyrosine residues analysed by western blots. (C) Western blot analysis of total protein extracts of E15.5 primary embryonic hepatocytes derived from Del-R26 Met embryos showing impaired phosphorylation of Met and Gab1 in the presence of the Met inhibitor PHA665752 (PHA; 1μM).

Article Snippet: Antibodies used were from Cell Signaling: anti-Met 25H2 (1:2000 for WB), anti-phospho-Tyr 1234/1235 -Met (1:2000 for WB; 1:50 for IHC), anti-phospho-Tyr 1003 -Met (1:2000 for WB), anti-phospho-Tyr 1349 -Met (1:1000 for WB), anti-phospho-Tyr 627 -Gab1 (1:2000 for WB), anti-phospho-Ser 473 -Akt (1:2000 for WB; 1:20 for IHC), anti-phospho-Ser 727 -Stat3 (1:2000 for WB), anti-phospho-Thr 202 -Tyr 204 -ERKs (#9106; 1:10000 for WB), anti-phospho-Thr 202 -Tyr 204 -ERKs (#4376; 1:150 for IHC), anti-ERKs (1:10000 for WB); from Santa-Cruz Biotechnology: anti-mouse Met (1:200 for WB), anti-human Met (1:1000 for WB), anti-HGF (1:500 for WB); from Abcam: anti-β-galactosidase (1:2000 for WB); from R&D: anti-mouseHGF (1:50 for IHC); from Sigma-Aldrich: anti-actin (1:12000 for WB); from Assay Designs: anti-human Met (1:500 for IHC); from hybridoma bank: Pax3 (1:10 for IHC), anti-myosin heavy chain II (MF20; 1:50 for IHC); from Jackson: anti-rabbit IgG-peroxidase or anti-mouse IgG-peroxidase (1:4000 for WB), anti-mouse fluorescent-coupled secondary antibodies (1:400 for IHC), anti-mouse or rabbit biotin-coupled secondary antibodies (1:500 for IHC).

Techniques: Western Blot, Derivative Assay, Expressing, Transgenic Assay

Journal: PLoS Genetics

Article Title: Tissue-Specific Gain of RTK Signalling Uncovers Selective Cell Vulnerability during Embryogenesis

doi: 10.1371/journal.pgen.1005533

Figure Lengend Snippet: (A) Whole mount ISH of E10.5 control and Del-R26 Met embryos with Hgf probe showing comparable expression domain in developing forelimbs. (B) Quantification analyses of Hgf 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: control, n = 6; Del-R26 Met , n = 6. Note no significant changes of signal in controls and Del-R26 Met mutants. (C) Immunofluorescence analysis of HGF (red) and Pax3 (green) proteins in transversal sections of E10.5 control and Del-R26 Met embryos at forelimb levels. Note the impaired myoblast migration (Pax3-positive cells) in Del-R26 Met mutants, whereas no significant changes are detected in HGF protein levels. Asterisks indicate non-specific staining in blood cells. Scale: 100μm. (D) Western blot analyses of total protein extracts of E10.5 control and Del-R26 Met dissected forelimbs. Note comparable levels of uncleaved HGF in Del-R26 Met mutants despite the ectopic expression of Met tg and the enhanced phosphorylation levels of Met and Gab1. (E) qRT-PCR analysis showing comparable Plau transcript levels in E10.5 dissected forelimbs of control (ctrl; n = 11) and Del-R26 Met (Del-Met; n = 11) embryos. (F) Western blot analysis of total protein extracts of E10.5 forelimb mesenchymal cell cultures showing phosphorylation levels of Met, Gab1, Akt, and ERKs in control and Del-R26 Met cultures, in cells untreated, stimulated with HGF (H) or in the presence of anti-HGF blocking antibodies (αH).

Article Snippet: Antibodies used were from Cell Signaling: anti-Met 25H2 (1:2000 for WB), anti-phospho-Tyr 1234/1235 -Met (1:2000 for WB; 1:50 for IHC), anti-phospho-Tyr 1003 -Met (1:2000 for WB), anti-phospho-Tyr 1349 -Met (1:1000 for WB), anti-phospho-Tyr 627 -Gab1 (1:2000 for WB), anti-phospho-Ser 473 -Akt (1:2000 for WB; 1:20 for IHC), anti-phospho-Ser 727 -Stat3 (1:2000 for WB), anti-phospho-Thr 202 -Tyr 204 -ERKs (#9106; 1:10000 for WB), anti-phospho-Thr 202 -Tyr 204 -ERKs (#4376; 1:150 for IHC), anti-ERKs (1:10000 for WB); from Santa-Cruz Biotechnology: anti-mouse Met (1:200 for WB), anti-human Met (1:1000 for WB), anti-HGF (1:500 for WB); from Abcam: anti-β-galactosidase (1:2000 for WB); from R&D: anti-mouseHGF (1:50 for IHC); from Sigma-Aldrich: anti-actin (1:12000 for WB); from Assay Designs: anti-human Met (1:500 for IHC); from hybridoma bank: Pax3 (1:10 for IHC), anti-myosin heavy chain II (MF20; 1:50 for IHC); from Jackson: anti-rabbit IgG-peroxidase or anti-mouse IgG-peroxidase (1:4000 for WB), anti-mouse fluorescent-coupled secondary antibodies (1:400 for IHC), anti-mouse or rabbit biotin-coupled secondary antibodies (1:500 for IHC).

Techniques: Expressing, Immunofluorescence, Migration, Staining, Western Blot, Quantitative RT-PCR, Blocking Assay

Journal: PLoS Genetics

Article Title: Tissue-Specific Gain of RTK Signalling Uncovers Selective Cell Vulnerability during Embryogenesis

doi: 10.1371/journal.pgen.1005533

Figure Lengend Snippet: (A) Scheme illustrating the experimental procedure employed for evaluating through MDCK cell scattering the bioavailability of HGF from control and Del-R26 Met 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 ), 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 ). (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 Del-R26 Met limb mesenchymal cells. Note a drastic reduction in the scattering response when MDCK cells are co-cultured with Del-R26 Met mutant cells (control: n = 4; Del-R26 Met : n = 3). Mann-Whitney and Student- t test.

Article Snippet: Antibodies used were from Cell Signaling: anti-Met 25H2 (1:2000 for WB), anti-phospho-Tyr 1234/1235 -Met (1:2000 for WB; 1:50 for IHC), anti-phospho-Tyr 1003 -Met (1:2000 for WB), anti-phospho-Tyr 1349 -Met (1:1000 for WB), anti-phospho-Tyr 627 -Gab1 (1:2000 for WB), anti-phospho-Ser 473 -Akt (1:2000 for WB; 1:20 for IHC), anti-phospho-Ser 727 -Stat3 (1:2000 for WB), anti-phospho-Thr 202 -Tyr 204 -ERKs (#9106; 1:10000 for WB), anti-phospho-Thr 202 -Tyr 204 -ERKs (#4376; 1:150 for IHC), anti-ERKs (1:10000 for WB); from Santa-Cruz Biotechnology: anti-mouse Met (1:200 for WB), anti-human Met (1:1000 for WB), anti-HGF (1:500 for WB); from Abcam: anti-β-galactosidase (1:2000 for WB); from R&D: anti-mouseHGF (1:50 for IHC); from Sigma-Aldrich: anti-actin (1:12000 for WB); from Assay Designs: anti-human Met (1:500 for IHC); from hybridoma bank: Pax3 (1:10 for IHC), anti-myosin heavy chain II (MF20; 1:50 for IHC); from Jackson: anti-rabbit IgG-peroxidase or anti-mouse IgG-peroxidase (1:4000 for WB), anti-mouse fluorescent-coupled secondary antibodies (1:400 for IHC), anti-mouse or rabbit biotin-coupled secondary antibodies (1:500 for IHC).

Techniques: Mutagenesis, Blocking Assay, Cell Culture, MANN-WHITNEY

Journal: Journal of Immunology Research

Article Title: Umbilical Cord-Derived Mesenchymal Stem Cells Inhibit Cadherin-11 Expression by Fibroblast-Like Synoviocytes in Rheumatoid Arthritis

doi: 10.1155/2015/137695

Figure Lengend Snippet: Suppression of soluble factors prevented the inhibitory effect of UCMSC on CDH11 expression by FLS from RA patients. Expression of CDH11 by FLS from RA patients, cocultured with UCMSC in the presence of anti-IL-10, anti-HGF antibody, or 1-MT. MSC: mesenchymal stem cells; FLS: fibroblast-like synoviocytes; CDH11: cadherin-11. ∗ P < 0.05; ∗∗ P < 0.01.

Article Snippet: Selected soluble factors inhibitors such as 1-MT, or an anti-IL-10 or anti-HGF antibodies (R&D Systems, Minneapolis, USA) were added to the coculture system.

Techniques: Expressing