Figure 1. Secretome of senescent fibroblasts enhances the migration of squamous cell carcinoma lines.

Cell motility was evaluated using the Transwell^® chamber migration assay. The lower compartments of the chamber were loaded with conditioned medium (CM) of senescent (SEN) and corresponding young (YNG) fibroblasts and incubated for 14 h at 37°C. DMEM containing 10% fetal bovine serum (FBS) and collagen type I at a concentration of 50 μg/ml served as positive controls. DMEM served as a negative control. Cells stained purple by Diff-Quick staining kit and counted under light microscopy at ×100 magnification (A) Representative images (×200 magnification, scale bars = 50 μm) showing migrated SCL-1 cells (pointed with red arrows) at the downside of the membrane in response to CM from senescent vs. young fibroblasts. Quantifications of cell migration proved that (B) Senescent (SEN) conditioned media of human dermal fibroblast strains FF95, FFRa and FFPia enhanced the migration of SCL-1 tumor cells compared with young (YNG) counterparts. Data are shown as mean ± S.D for n = 3 replicates. ***p < 0.001 calculated by unpaired student t-test between SEN CM-treated and YNG CM- treated groups for each fibroblast strains. (C) Conditioned media of FF95 senescent fibroblasts increased the migration of all tested tumor cells (SCL-1, SCC-12B2, SCC-13 and A431) but had no effect on normal keratinocytes. Data are shown as mean ± S.D for n = 3 replicates; Graphs represent one of the three independent experiments; *p < 0.05, **p < 0.01 and ***p < 0.001 calculated by unpaired student t-test between SEN CM-treated and YNG CM- treated groups for each cell line; HPF = ×100 magnification. (Note that due to low standard deviations of some measurements, error bars are not visible for all data points.).