siRNA knockdown of endogenously expressed Tensin3 in human melanoma cells increases cell migration.

Endogenous Tensin3 expression was knocked down over 24 h in human melanoma cells WM793 using three different Tensin3 siRNA constructs (siRNA I, II, III), as well as non-silencing siRNA and transfection mixture only (negative controls). (A) mRNA level of Tensin3 in WM793 cells after 24 h gene silencing, analyzed by qRT-PCR. Results of gene expression were normalized to endogenous control gene (B2M) and are presented as Tensin3 (TNS3) expression relative to non-silenced control (transfection mix only; first bar). Bars shown are means of duplicate determinations from a representative experiment of four. (B) Protein level of Tensin3 (TNS3) in WM793 cells after 24 h gene silencing, analyzed by western blot. Whole cell lysates were subjected to 8% SDS-PAGE and western blot detection with anti-Tensin3 antibody. Band intensities were quantified by densitometry and normalized to GAPDH protein level on the same blot. Bars are Tensin3 protein expression relative to non-silenced control (transfection mix only; first bar). (C) Migration of WM793 cells over 16 h, treated with different siRNA constructs as well as negative controls (transfection mix only, and non-silencing siRNA). Each bar represents mean±SEM migrated cells expressed as fold difference relative to negative control cells (transfection mix only) (n = 3 separate experiments, duplicate inserts). ***P<0.001 control vs. Tensin3 siRNA II; **P<0.01 control vs. Tensin3 siRNA I and III; no significant difference for control v non-silencing siRNA (ANOVA with Bonferroni adjustment).