FIGURE 6.
Knockdown of ERK3 promotes melanoma cells migration. Transwell migration assays were performed 2 days posttransfection with siERK3 in (a) BRAFV600E human melanoma cell lines A375, and (b) human WM3211 BRAF WT melanoma cell lines, or using (c) A375 by shRNA stably depleted using a lentiviral system targeting the ERK3 coding region with shGIPZ as empty vehicle control. Transwell migration was consistent with transient knockdowns. Knockdown efficiencies were verified by western blots. Migration was quantified by two-chamber transwell migration assays. Migrated cell numbers were quantified from an average of six captured fields (×20 magnification) per insert for two technical replicates per biological replicate (n > 3) after 8–12 hr depending on the cell line. Presented captures were from the replicate with cell number closest to the average. Significance levels (p values) were determined by Student’s t test and expressed as mean ± standard deviation. (d) Stable shERK3 A375 were used for scratch healing assays. Healing distances were measured under a ×4 magnification microscope field with six wells per cell line after 24 hr. Presented captures were from the one with cell number most close to the average. Significance levels (p values) were determined by Student’s t test and expressed as mean ± standard deviation (n = 6). BRAF: v-Raf murine sarcoma viral oncogene homolog B; ERK3: extracellular signal-regulated kinase 3; siERK3: ERK3 knockdown