Figure 1. PFKFB4 controls in vitro cell migration in metastatic melanoma in a glycolysis-independent manner.

(A, B) Quantification of PFKFB4 mRNA and protein levels in four human melanoma cell lines: MNT1, A375M, MeWo, and Lu1205. (A) Relative PFKFB4 mRNA levels measured by RT-qPCR and normalized by the expression of 18S and TBP housekeeping genes. Error bars: SEM. (B) PFKFB4 protein levels detected by Western-blotting, with PFKFB4/actin relative quantification (optical density). (C) PFKFB4 protein levels in MeWo and A375M cells 48 h after transfection with siRNA targeting PFKFB4 with or without co-transfection with Xenopus laevis PFKFB4 plasmid. (D) Starting 48 h after transfection, cell migration was tracked for 16 h from phase-contrast images. Scale bar is 20 μm. Each point corresponds to the average speed of one cell. (E, F, G, H, I) MeWo (E, G, H, I) or A375M (F) cells were co-transfected either with siControl/empty plasmid, siPFKFB4/empty plasmid, or with siPFKFB4 together with a X. laevis PFKFB4 plasmid in its wild-type form (E, F, I) or mutant forms (I). 27 independent biological replicates were performed, with 50–100 cells counted in each condition. Velocity reduction was in average of 33% for MeWo cells and of 42% for A375M cells. (G, H) The average speed was also measured when cells were cultured in glucose-free medium (G) or complete medium supplemented with 2DG (H). In each panel, a representative experiment is shown (n > 3), and displays mean ± SEM. P-values were calculated using the Mann–Whitney test. n.s.: P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

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