Fig. 4

AMG-based treatment triggers matrix metalloproteinase expression and enzymatic activity in murine primary fibroblasts. a Volcano plot showing up- (red dots) and down-regulated (light blue dots) genes altered by AMG treatment (5 h of stimulation). Gene values are reported as a Log₂FoldChange (p value < 0.05). b MMP gene expression evaluated by RT-qPCR. Expression values are expressed as a -∆CT normalized on the expression of Gapdh, B-actin, and Rpl13a housekeeping genes (N = 3). Differences are calculated with multiple unpaired t test (N = 3) and indicated as *P < 0.05. c Enzymatic activity of MMP members present in cell supernatant was fluorometrically detected. Data are presented as Relative Fluorescence Units (RFU). Signals were evaluated 30 min after starting the reaction using a microplate reader with a filter set of Ex/Em = 485/535. The fluorescence signal obtained from each sample was normalized on the substrate control. Groups of wounded cells that did not receive any treatment were used as a control. d Representative images of scratch wound assays on monolayers of murine primary fibroblasts. AMG was applied on wounded cells with different conditions (unprocessed AMG and its soluble fraction) and for different time periods (1, 5, and 12 h). Each condition was also incubated with the MMPs inhibitor—Actinonin—20 μM for the same time periods of AMG treatment. Images were taken at the beginning (T0) and at regular intervals until wound closure was achieved. Scale bar = 200 μm. e Quantifications of AMG-treated cells with or without Actinonin are reported. Data are presented as mean ± SEM. Differences were calculated with multiple unpaired t test (N = 3) and indicated as *P < 0.05