Figure 3. ICMT and PFKFB4 both control RAS addressing at the plasma membrane and melanoma cell migration.

(A, B, C) Average speed of MeWo cells transfected with siRNA (siCtrl, siPFKFB4, or siICMT) and plasmid (empty vector, hICMT-myc-Flag, hPFKFB4-myc-Flag, or RasV12). (A, B, C) Graphs show the mean calculated in one experiment with at least 30 cells in each condition (A: n = 1, B: n = 1, C: n = 3), Error bars are calculated with SEM. P-values are calculated using the Mann–Whitney test. n.s.: P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; and ****P < 0.0001. (D) Detection of RAS subcellular localization by immunostaining on MeWo cell transfected with RasV12-HA (yellow). Nuclei were stained with DAPI (blue). At the plasma membrane, RAS was found distributed according to three main phenotypes: either a clear and homogeneous membrane localization (Phenotype 1), or the absence of signal (Phenotype 3), or an intermediate phenotype with intermittent RAS expression at the membrane (Phenotype 2). Insets show enlargements of the areas framed in red. Scale bar is 10 μm. The proportion of each phenotypes was quantified after transfection of either siControl (nbcell = 43), or siICMT (nbcell = 51), or siPFKFB4 (nbcell = 46). A representative experiment is shown, n = 3.

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