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