Journal: Developmental biology

Article Title: Myostatin-deficient medaka exhibit a double-muscling phenotype with hyperplasia and hypertrophy, which occur sequentially during post-hatch development.

doi: 10.1016/j.ydbio.2011.08.027

Figure Lengend Snippet: Fig. 2. Generation of mstnC315Y medaka. (A) Sequence data for each genotype. Lines in green, red, black and blue indicate adenine, thymine, guanine and cytosine, respectively. Three-letter amino-acid codes show the resulting transcript. Top figure illustrates WT medaka sequence, while middle and bottom figures illustrate heterozygous and homozygous C315Y mutation, respectively. Guanine to adenine nucleotide transition causes an amino-acid change from Cys to Tyr at amino acid 315 (black triangles). (B) Demonstration of de- ficient medaka for the MSTN signaling pathway. Phosphorylated Smad2 in nuclear proteins were targeted and detected with western blotting. Nucleus protein were obtained from muscles of WT (left lane) and mstnC315Y (middle lane) at 16 wk post-hatching. Upper figure shows decreased expression of phosphorylated Smad2 in mstnC315Y. TGF-β treated Mv1Lu cell lysates were used as positive control of phosphorylated Smad2 for western blotting (right lane). Lower figure shows histone H2B used as a loading control.

Article Snippet: TGF-β treated Mv1Lu cell lysates (Santa Cruz Biotechnology, Inc., CA, USA) were used as a positive control for phosphorylated Smad2 protein.

Techniques: Sequencing, Mutagenesis, Western Blot, Muscles, Expressing, Positive Control, Control

Journal: The Journal of biological chemistry

Article Title: c-Src kinase activity is required for hepatocyte growth factor-induced motility and anchorage-independent growth of mammary carcinoma cells.

doi: 10.1074/jbc.273.50.33714

Figure Lengend Snippet: FIG. 1. c-Src kinase activity is elevated in SP1 carcinoma cells compared with Mv1Lu epithelial cells. Cell lysates were prepared from serum-starved Mv1Lu and SP1 cells treated without (2) or with (1) HGF (40 ng/ml) for 10 min and were immunoprecipitated with anti-c-Src IgG. Immunoprecipitates were subjected to an in vitro kinase assay using enolase as a substrate, and kinase activity was measured as described under “Experimental Procedures.” A, autoradiogram show- ing 32P-labeled enolase. B, quantitation of autoradiogram using Phos- phorImager. Results are expressed as the percentage of cpm in un- treated Mv1Lu cells (100%), normalized to the amount of c-Src protein in C. The means 6 range of two experiments are shown. Similar results were obtained using the c-Src kinase family-specific cdc2 peptide as substrate (data not shown). C, Western blot analysis of immmunopre- cipitates in A, probed with anti-c-Src IgG.

Article Snippet: Briefly, lysates from SP1 and Mv1Lu cells were prepared, and equal protein amounts from each cell lysate were immunoprecipitated with anti-c-Src IgG (Santa Cruz Biotechnology) as described above.

Techniques: Activity Assay, Immunoprecipitation, In Vitro, Kinase Assay, Labeling, Quantitation Assay, Western Blot

Journal: The Journal of biological chemistry

Article Title: c-Src kinase activity is required for hepatocyte growth factor-induced motility and anchorage-independent growth of mammary carcinoma cells.

doi: 10.1074/jbc.273.50.33714

Figure Lengend Snippet: FIG. 2. c-Src kinase binds to tyrosine-phosphorylated Met. Cell lysates derived from serum-starved Mv1Lu cells treated without (2) or with (1) HGF (40 ng/ml) for 15 min were immunoprecipitated with anti-c-Src IgG (A) or anti-Met IgG (B). The immune complexes were separated by 8% SDS-PAGE and immunoblotted with anti-Met IgG (A) or anti-c-Src IgG (B). Protein molecular mass standards are shown on the right. This experiment was done twice with similar results.

Article Snippet: Briefly, lysates from SP1 and Mv1Lu cells were prepared, and equal protein amounts from each cell lysate were immunoprecipitated with anti-c-Src IgG (Santa Cruz Biotechnology) as described above.

Techniques: Derivative Assay, Immunoprecipitation, SDS Page

Journal: The Journal of biological chemistry

Article Title: c-Src kinase activity is required for hepatocyte growth factor-induced motility and anchorage-independent growth of mammary carcinoma cells.

doi: 10.1074/jbc.273.50.33714

Figure Lengend Snippet: FIG. 5. Expression of dominant negative mutant SRC-RF does not alter Met protein levels or activity and downstream signal- ing. A, SP1 cells transfected with SRC-RF or SRC or untreated SP1 cells were prestarved overnight and lysed as described in the legend to Fig. 1. Equal amounts of protein from each lysate were concentrated on Microcon 10 filters (Amicon Inc., Beverly, MA) and analyzed by Western blotting with anti-Met IgG (top panel). The blot was stripped and reprobed with anti-phosphotyrosine antibody (middle panel). Cell ly- sates were also immunoprecipitated with anti-Met IgG, and immuno- precipitates were subjected to an in vitro Met kinase assay as described under “Experimental Procedures.” The autoradiogram depicting 32P- labeling of Met is shown (bottom panel). Relative band intensities and amount of 32P labeling was determined using a Storm PhosphorImager. The relative amount of Met tyrosine phosphorylation (1.0, 1.0, or 1.0) or of in vitro Met autophosphorylation (1.0, 1.1, or 1.0) was not signifi- cantly different among the three cell lines. B, serum-starved SP1 cells transfected with SRC-RF or SRC and untreated SP1 cells were lysed as described in the legend to Fig. 1. Prestarved Mv1Lu cells untreated or treated with HGF (40 ng/ml) for 10 min were used as negative and positive controls, respectively. Equal amounts of protein from each lysate were immunoprecipitated with anti-PCL-g1 IgG. Immunopre- cipitates were subjected to 7% SDS-PAGE and transferred to nitrocel- lulose. The blot was probed with anti-PCL-g1 IgG (top panel) before being stripped and reprobed with anti-phosphotyrosine antibody (bot- tom panel). This experiment was done twice with similar results. IP, immunoprecipitation; IB, immunoblot.

Article Snippet: Briefly, lysates from SP1 and Mv1Lu cells were prepared, and equal protein amounts from each cell lysate were immunoprecipitated with anti-c-Src IgG (Santa Cruz Biotechnology) as described above.

Techniques: Expressing, Dominant Negative Mutation, Activity Assay, Transfection, Western Blot, Immunoprecipitation, In Vitro, Kinase Assay, Labeling, Phospho-proteomics, SDS Page

Journal: Scientific Reports

Article Title: Oleanolic acid stimulation of cell migration involves a biphasic signaling mechanism

doi: 10.1038/s41598-022-17553-w

Figure Lengend Snippet: C-Jun and EGFR OA activation, necessary for OA-cell migration, are decoupled in Mv1Lu cells. ( a ) The figure shows representative images of cell migration obtained under control conditions compared to those with 5 µM OA and OA plus 2,5 µM EGFRi after 24 h treatment. Scale bar 200 µm. ( b ) Plot represents cell migration as the difference obtained between the quantified areas at time 0 h and time 24 h in each condition. Asterisks indicate statistically significant differences between the selected conditions according to a one-way ANOVA statistical analysis: (*p < 0.05, **p < 0.005, ***p < 0.001 and ****p < 0.0001). ( c ) Total protein extracts from serum-starved sub-confluent Mv1Lu cells treated with 5 µM OA, EGF or DMSO equivalent volume as vehicle control. Different protein phosphorylation was assayed at the indicated times (h) targeting phospho-EGFR (Tyr 1068), phospho-ERK1/2 (Thr 202/Tyr 204), phospho-JNK1/2 (Thr 183/Tyr 185) and phospho-c-Jun (Ser 63). Additionally, total protein expression was assayed: ERK1/2, JNK1/2 and c-Jun. β-Actin was used as a loading control. A representative experiment is shown. ( d ) Plots with intensity values of each protein assayed by Western-blot, by gathering the data of three independent experiments. Asterisks indicate statistically significant differences between the selected conditions according to a one-way ANOVA statistical analysis: (*p < 0.05, **p < 0.005, ***p < 0.001 and ****p < 0.0001).

Article Snippet: Mink Lung Epithelial (Mv1Lu) , , cells were grown in Eagle's Minimum Essential Medium (EMEM) (Biowest, Nuaillé, France).

Techniques: Activation Assay, Migration, Control, Phospho-proteomics, Expressing, Western Blot

Journal: Scientific Reports

Article Title: Oleanolic acid stimulation of cell migration involves a biphasic signaling mechanism

doi: 10.1038/s41598-022-17553-w

Figure Lengend Snippet: Specific inhibitors against EGFR, MEK and JNK, upon OA treatment, show the independent OA-activation on c-Jun at the edge of wound-scratched Mv1Lu cells at wound edge. ( a ) Confluent Mv1Lu cells were scratched and allowed to migrate for 6 h. Cells treated with 5 µM OA or DMSO equivalent volume were immunostained with specific antibodies against c-Jun transcription factor. Additionally, cells were treated 30 min before with the following inhibitors: 2.5 µM EGFRi, 50 µM MEKi and 15 µM JNKi. Co-staining with phalloidin and Hoechst-33258 was used to show actin cytoskeleton and nuclei, respectively. Images of p-c-Jun fluorescence were converted into pseudo-color with ImageJ software to show the intensity of c-Jun staining. Color rainbow scale represents fluorescence intensity for phospho-c-Jun. Actin fibers (F-actin): red. Nuclei: blue. Images were obtained with a confocal microscope. This experiment was repeated at least three times. Representative images are shown. Scale bar indicates 10 µm. ( b ) Plot represents the data obtained by p-c-Jun intensity at cell nuclei. In every condition, each point on the plot represents p-c-Jun intensity at the nucleus of one cell, quantified by ImageJ software. With the collected data of p-c-Jun intensity, a one-way ANOVA statistical analysis was performed (*p < 0.05, **p < 0.005, ***p < 0.001 and ****p < 0.0001).

Article Snippet: Mink Lung Epithelial (Mv1Lu) , , cells were grown in Eagle's Minimum Essential Medium (EMEM) (Biowest, Nuaillé, France).

Techniques: Activation Assay, Staining, Fluorescence, Software, Microscopy

Journal: Scientific Reports

Article Title: Oleanolic acid stimulation of cell migration involves a biphasic signaling mechanism

doi: 10.1038/s41598-022-17553-w

Figure Lengend Snippet: OA promotes changes in FAs and actin cytoskeleton. Confluent Mv1Lu cells were scratched and allowed to migrate for 6 and 12 h. Cells treated with 5 µM OA or DMSO equivalent volume (vehicle control) were immunostained with specific antibodies against paxillin (green). Co-staining with phalloidin and Hoechst-33258 was used to show actin cytoskeleton (magenta) and nuclei (blue), respectively. This experiment was repeated at least three times. Representative images are shown. Scale bar indicates 10 µm. Quantification of the density of FA as FA number per filopodia area. Quantification of FA size (average size) at the filopodia area. One-way ANOVA statistical analysis was performed (*p < 0.05, **p < 0.005, ***p < 0.001 and ****p < 0.0001).

Article Snippet: Mink Lung Epithelial (Mv1Lu) , , cells were grown in Eagle's Minimum Essential Medium (EMEM) (Biowest, Nuaillé, France).

Techniques: Control, Staining

Journal: Scientific Reports

Article Title: Oleanolic acid stimulation of cell migration involves a biphasic signaling mechanism

doi: 10.1038/s41598-022-17553-w

Figure Lengend Snippet: OA induces FAK phosphorylation and promotes its localization in focal adhesion with paxillin in Mv1Lu cells. ( a ) The activation of FAK was assessed by Western Blot at Tyr 925, at the indicated times in the presence of 5 µM OA, DMSO equivalent volume or EGF. Additionally, total protein expression was assayed: FAK. β-Actin was used as a loading control. Phospho-FAK intensity values in Western-blot were quantified by ImageJ to conduct a one-way ANOVA analysis showed on the plot (*p < 0.05, **p < 0.005, ***p < 0.001 and ****p < 0.0001). ( b ) Confluent Mv1Lu cells were scratched and allowed to migrate for 6 h. Pictures show the immunostaining with specific antibodies against phospho-FAK (Tyr 925) and paxillin. Co-staining with Hoechst-33258 was used to reveal nuclei. P-FAK: green. Paxillin: red. Nuclei: blue. ( c ) Pictures and graphs represent a colocalization analysis performed by Zeiss Efficient Navigation (ZEN) software. Colocalized pixels are highlighted in white at the immunostaining images. Graphs are dot plots representing both p-FAK and paxillin intensities in terms of number of pixels: p-FAK pixels on Y axis and paxillin pixels on X axis. The 3 quadrant (upper right) represents overlapped (common) pixels between both proteins. ( d ) Pictures of each condition (two) were divided in three horizontally distributed sectors. Pearson’s correlation coefficient in each sector of each condition was calculated by the average pixel intensity of p-FAK and paxillin overlapped pixels. The plot represents Pearson’s correlation values for each condition, each dot representing the value obtained in one sector. Asterisks indicate statistically significant differences between conditions according to one-way ANOVA statistical analysis: (*p < 0.05, **p < 0.005, ***p < 0.001 and ****p < 0.0001). This experiment was repeated at least three times. Representative images are shown. Scale bar indicates 10 µm.

Article Snippet: Mink Lung Epithelial (Mv1Lu) , , cells were grown in Eagle's Minimum Essential Medium (EMEM) (Biowest, Nuaillé, France).

Techniques: Phospho-proteomics, Activation Assay, Western Blot, Expressing, Control, Immunostaining, Staining, Software

Journal: Scientific Reports

Article Title: Oleanolic acid stimulation of cell migration involves a biphasic signaling mechanism

doi: 10.1038/s41598-022-17553-w

Figure Lengend Snippet: EGFR and MEK inhibitors prevent OA-induced FAK localization at FA in Mv1Lu cells. ( a ) Confluent Mv1Lu cells were scratched and allowed to migrate for 6 h. Cells were treated 30 min before scratch and subsequent OA treatment with specific inhibitors 2,5 µM EGFRi, 50 µM MEKi and 15 µM JNKi. Pictures show the immunostaining with specific antibodies against phospho-FAK (Tyr 925) and paxillin. Co-staining with Hoechst-33258 was used to reveal nuclei. ( b ) The plot represents a colocalization analysis performed by Zeiss Efficient Navigation (ZEN) software. Pictures of each condition (two) were divided in three horizontally distributed sectors. Pearson’s correlation coefficient in each sector of each condition was calculated by the average pixel intensity of p-FAK and paxillin overlapped pixels. The plot represents Pearson’s correlation values for each condition, each dot representing the value obtained in one sector. Asterisks indicate statistically significant differences between conditions according to a one-way ANOVA statistical analysis: (*p < 0.05, **p < 0.005, ***p < 0.001 and ****p < 0.0001). This experiment was repeated at least three times. Representative images are shown. Scale bar indicates 10 µm.

Article Snippet: Mink Lung Epithelial (Mv1Lu) , , cells were grown in Eagle's Minimum Essential Medium (EMEM) (Biowest, Nuaillé, France).

Techniques: Immunostaining, Staining, Software