Figure 6.

Histone deacetylase inhibitor treatment strongly decreases the migratory activity of A375 melanoma cells in a PMCA4b-dependent manner. A375 cells were treated with 4 mM valproate for 48 h before the migration analysis. (A) The random migratory activity of A375 melanoma cells was analyzed by automated fluorescence microscopy for 24 h (A1). Single cell migration trajectories of untreated, control cells (left graph) and valproate-treated cells (right graph) were recorded. Starting position of each cell trajectory was fixed to the origin of the plot. (A2) Mean velocity was evaluated for 24 h of migration. Data are shown as mean ± SEM of ≥100 individual cells of at least three independent measurements. Significance compared to control is denoted by ***p < 0.001; two-tailed unpaired t-test. (B,C). Effect of valproate treatment of A375 cells (B) and specific inhibition of PMCA4 with caloxin 1c2 (C) on the directional migration of A375 cells. The effect of PMCA4 inhibition was studied on untreated (C1) and 4 mM valproate-treated (C2) A375 cells. The directional migration activity was assessed using Boyden chambers. B1 images show representative fields of view from the bottom of the filter (control and 4 mM valproate-treated A375 cells). B2 shows migration of cells in response to different concentrations (1–5 mM) of valproate. The cells that migrated through the membrane to the bottom face were counted. The bar graphs (B2, C1, C2) indicate the average number of migrated cells per field. Data are mean of three experiments ± SEM. Significance of comparisons is denoted by ***p < 0.001; two-tailed unpaired t-test.