Figure 2. ANGPTL4 controls the malignancy phenotype of cutaneous and brain metastasizing melanoma variants.

A.-C. Cutaneous (CUT) and melanoma brain metastasizing (MBM) variants were transduced with an ANGPTL4 cDNA-containing construct (ANGPTL4^hi) or with the backbone construct pQCXIP (CON^pQC). MBM cells were transduced with a mixture of 4 different shANGPTL4-containing constructs (ANGPTL4^lo), or with the control construct (CON^sh). The efficacy of ANGPTL4 over-expression or down-regulation was verified: (A) RT-qPCR analysis was performed to determine the mRNA expression level of ANGPTL4. The bars represent the relative ANGPTL4 expression (normalized to RS9) in ANGPTL4^hi or ANGPTL4^lo cells compared to control cells + SEM obtained in one measurement in at least three independent experiments. *P < 0.05. (B, C) Supernatants of the transduced cells were subjected to Western blot analysis. ANGPTL4 was detected by specific Abs. The bars represent the relative ANGPTL4 expression in ANGPTL4^hi or ANGPTL4^lo cells compared to control cells + SEM obtained in one measurement in two-three independent experiments. *P < 0.05. Representative blots are shown. Ponceau staining was used for loading control. D., E. Melanoma cells were allowed to migrate through collagen coated transwells for 24 hrs. The migrated cells were fixed and counted. (D) Representative images are presented (X10 magnification). (E) The bars represent the average number of migrating cells per field in three independent experiments performed in triplicates + SEM. *P < 0.05. F. BEC were cultured for 24 hrs to form a confluent monolayer and stimulated with 100 units/ml TNFα and IFNγ for additional 24 hrs. mCherry- or GFP-expressing melanoma cells were added and allowed to adhere for 30 min at 37°C. The fluorescence signal of labeled cells was measured before and after removal of non-adherent cells. The bars represent the average % adherent cells (normalized to control cells) + SEM in at least three independent experiments. Six replicates were performed in each experiment. *P < 0.05. G.-I. BEC were cultured for 48 hrs on the upper side of the apical chamber of transwell inserts to form a confluent monolayer. Astrocytes were cultured in 24-well plates for 24 hrs and starved for additional 24 hrs to allow the secretion of soluble factors. mCherry-expressing melanoma cells were added onto the BEC monolayer and allowed to migrate for 24 hrs towards the astrocytes (G). The migrated cells were fixed and counted. (H) Representative images are presented (X10 magnification). (I) The bars represent the average number of migrating cells per field (normalized to control cells) in at least three independent experiments performed in triplicates + SEM. *P < 0.05.