Fig. 3
Functional and morphological effects of LAD1 knockdown in OSCC cell lines. (a) Cell survival analysis by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2 H-tetrazolium, inner salt (MTS) assay. (b) Collagen gel invasion assay. (c) Transwell migration assay. (d) Scratch wound-healing assay. (e) Cell-tracking plots of timelapse imaging for 24 h and comparisons of velocity and distance/frame; scale bars: 100 μm (b, c). Bars in bar charts, means ± s.e.; *p < 0.05, **p < 0.01. In MTS assay (a), three cell lines showed suppression of cell survival rates in si#1 treatments. HSC-4 showed the common suppressive effect of LAD1 knockdown on cell survival. In the collagen gel invasion assay, depths of cell infiltration, i.e., absolute length from the uppermost collagen surface to the deeper cancer cells in captured foci were measured in the vertical sections of paraffin-embedded culture samples. In si#1 LAD1knockdown of the three cell lines, the cancer cells showed considerable deep infiltration (b). HSC-2 and HSC-4 with si#2 and HSC-2 and HSC-3 with si#3 also showed increased depth of infiltration. In transwell migration assays, si#1 and si#3 effectively suppressed cell migration 24 h after seeding, whereas si#2 did not (c). In the wound scratch assay with serum-free media, LAD1-knockdown cells showed wider gaps than controls 48 h after scratching only in HSC-2 and HSC-4 with si#1 (d). The time-lapse analysis was performed to analyze cellular planer motility (e). The LAD1-knockdown cells showed decreased average velocity and moving distance between the time frames.
