siRNA-mediated downregulation of PKCα results in AMT in A375m2 amoeboid cells. A) Cell morphology analysis of A375m2 strain transfected with siRNA against PKCα. Cells were transfected with siRNA and their morphology was observed after 48 hours in 3D collagen. Cells were analyzed using NIS Elements software (Nikon). Cells were classified according to the elongation index as rounded, intermediate or elongated (see Methods section). Statistical significance (marked by asterisk) was evaluated using Pearson’s Chi-squared test. Representative result is shown out of three independent experiments. B) Cell invasion analysis of A375m2 strain transfected with siRNA against PKCα. Cells were transfected with siRNA and let to invade into 3D collagen for 48 hours. 3D collagen cross-sections were analyzed using NIS Elements software (Nikon) and average invasion depth was calculated and normalized to that of untreated cells The invasion depth in each individual field of view was assessed as sum of invasion depths of individual cells divided by the total number of cells in the respective field of view (including cells on the top of collagen). Statistical significance (marked by asterisks) was evaluated using ANOVA followed by Tukey’s honest significant difference test.