Fig. 4: PLX8394 inhibits RT3 cell invasion through collagen I.

See also Supplementary Fig. S4. A RT3 cells were treated with 10 μM PLX8394 for 24 h in 2D condition, followed by spheroid formation with skin primary fibroblasts. The spheroids were allowed to grow for 3 days, after which they were transferred to a 96-well plate and embedded with a collagen I gel. The invasion was followed by a confocal microscope every 24 h during 5 days. RT3 cells, red. Scale bar, 500 μm. From each time point, 2–4 spheroids were imaged and analyzed. Three independent biological replicates were performed. B RT3 cell invasion from cocultured spheroids that were treated as in (A). Left, a representative graph from three biological replicates. Right, analysis of RT3 cell invasion from cocultured spheroids. The graph shows the difference in RT3 cell invasion between DMSO treated control samples and PLX8394 treated samples. The graph shows mean from three independent biological replicates (squares) ± SD. (Each replicate contained 3-4 spheroids). The p values are from paired t-test. C RT3 cells were treated with 10 μM SB203580 for 24 h in 2D condition, followed by spheroid formation with skin primary fibroblasts. The spheroids were allowed to grow for 3 days, after which they were transferred to a 96-well plate and embedded with a collagen I gel. The invasion was followed by a confocal microscope every 24 h during 5 days. Left, a representative graph from three biological replicates. Right, analysis of RT3 cell invasion from cocultured spheroids. The graph shows difference in RT3 cell invasion between DMSO treated control samples and SB203580 treated samples. The graph shows mean from three independent biological replicates (squares) ± SD. (Each replicate contained 3-4 spheroids). The p value is from paired t-test. RT3 cells and cSCC cells were treated in 2D condition with PLX8394 (10 μM) for 24 h, followed by 3D spheroid formation with skin primary fibroblasts. The spheroids were allowed to grow for 3 days before harvesting for western blotting. MMP-1 (D and E) and MMP-13 (F) levels were analyzed by western blotting and β-actin was used as a loading control. Representative images from three independent biological replicates are shown. G RT3 cells were treated with 1 μM MMP Inhibitor III for 24 h in 2D condition, followed by spheroid formation with skin primary fibroblasts. The spheroids were allowed to grow for 3 days, after which they were transferred to a 96-well plate and embedded with a collagen I gel. The invasion was followed by a confocal microscope every 24 h during 5 days. Left, a representative graph from three biological replicates. Right, analysis of RT3 cell invasion from cocultured spheroids. The graph shows difference in RT3 cell invasion between DMSO treated control samples and MMP inhibitor treated samples. The graph shows mean from three independent biological replicates (squares) ± SD. (Each replicate contained 5–8 spheroids). The p values are from paired t-test.