Fig. 6

MG treatment promotes matrix remodeling through accelerated migration and enhanced MMP activity in keratinocytes. a Representative western blotting showing protein level of phosphorylated and total ERK1/ERK2 obtained from unwounded human keratinocytes upon 30 min, 1 and 5 h of MG treatment and in which we induced ERK inhibition (using the MEK inhibitor PD0325901—1 μM). b Representative images of MG-treated keratinocytes in a scratch wound assay. MG was applied for different time periods (5 and 12 h). Images were taken at the beginning (T0) and at regular intervals until closure was achieved. Each condition was also incubated with the MEK inhibitor—PD0325901—1 μM for the same time periods of the MG treatment. Wounded cells did not receive any treatment and were used as a control. Scale bar = 200 μm. c Quantifications of each time of treatment are reported in the graphs. Data are presented as mean ± SEM (standard error of the mean). Significant differences between control vs AMG are calculated with one-way ANOVA and indicated as *P < 0.05; **P < 0.01. Significant differences between AMG vs AMG + PD0325901 are calculated with one-way ANOVA and indicated as °P < 0.05; °°P < 0.01; °°°P < 0.005. Significant differences between control vs AMG + PD0325901 are calculated with one-way ANOVA and indicated as #P < 0.05; ##P < 0.01. (N = 3). d Observation of transwell chambers. Migrated cells were stained with crystal violet 0.1%, observed under a light microscope and analyzed using ImageJ software. Data were presented as mean ± SEM. Differences are calculated using one-way ANOVA (N = 4) and indicated as **P < 0.01. Scale bar = 100 μm. e Keratinocytes viability upon MG treatment was evaluated by flow cytometry using the fixable viability stain 660. Percentage of viable cells upon 5 and 12 h of AMG treatment are reported. Statistical analyses were performed using two-tailed unpaired t test. No significant differences were found between AMG-treated and untreated cells. (N = 3). f Ki67 staining analyses on AMG-treated keratinocytes (5 h and 12 h). Together with MG, the MEK inhibitor—PD0325901—was applied on human keratinocytes to assess ERK signaling involvement in cell proliferation. Untreated cells were used as controls. No differences in percentage of Ki67⁺ cells were evaluated in all the conditions under study. Data are presented as mean ± SEM. One-way ANOVA was used to perform statistical analysis (N = 3). g MMP gene expression evaluated by RT-qPCR in vehicle and 5 h AMG-treated in the presence or absence of PD0325901 1 μM. Expression values are expressed as a z-score of the average fold change (FC) normalized on the expression of GAPDH, B-ACTIN, and RPL13A housekeeping genes (N = 3). h MMP gene expression evaluated by RT-qPCR in vehicle and 12 h AMG-treated in the presence or absence of PD0325901 1 μM. Expression values are expressed as a z-score of the average fold change (FC) normalized on the expression of GAPDH, B-ACTIN, and RPL13A housekeeping genes (N = 3)