Figure 1

AnxA6 overexpression and pharmacological NPC1 inhibition reduce A431 cell migration. (a,b) A431-WT and A431-A6 cells were treated ± U18666A (4 µg/ml) and then wound healing assays were performed. Representative images at t = 0 and 24 h are shown. The relative wound density (RWD %) and speed of wound closure (%/h) from 3 independent experiments with triplicate samples (mean ± SD) is shown. (c) A431-WT treated ± U18666A (4 µg/ml), ectopically expressing NPC1 wildtype (NPC1-WT) or mutant (NPC1-P692S), and A431-A6 cells were grown until 90% confluency and then wound healing assays were performed. The RWD (%) at t = 18 h from 2 independent experiments with duplicate samples (mean ± SD) was calculated. (d) A431-WT stably expressing scrambled shRNA (scr) or a combination of four shRNAs (1–4) targeting NPC1 (NPC1-KD) were fixed and stained with filipin. Arrowheads indicate filipin staining at the plasma membrane of control cells. Arrows point at cholesterol accumulation in perinuclear compartments of NPC1-depleted A431-WT cells. The mean filipin intensity per cell (a.u.) in 32 control (scr) and 36 NPC1-KD cells was quantified (mean ± SD). Bar is 10 µm. (e–f) Wound healing assays with A431-WT stably expressing scrambled shRNA (scr) or NPC1-depleted A431-WT cells using the IncuCyte were performed. Representative images at t = 0 and 18 h are shown (e). Cell migration was monitored at 2 h intervals and RWD (%) and cell velocity (µm/h) from 2 independent experiments with quadruple (left panel) or quintuple samples (right panel) was calculated (mean ± SD). The mean of individual experiments (b,c) and individual data points (f, left panel) is indicated by dot points in each bar graph. *p < 0.05, **p < 0.01, ***p < 0.001; two-way ANOVA with Tukey’s post-hoc test (b,f), one-way ANOVA with Tukey’s post-hoc test (c) and Mann–Whitney two-tailed test (f).