Figure 3
From: CD271 determines migratory properties of melanoma cells
CD271 controls expression of mediators of cell migration. (A) Left panels: Absence of CD271 in melanoma cells (T20/02) stably transfected with a CD271-targeting shRNA (shCD271/sh#3) but high expression in control cells (shCtl.) as determined by immunofluorescence and a directly labeled (PE, red) CD271 antibody. Right panels: A changed morphology of CD271 knock-down cells is indicated by phalloidin (red) and bright field images (PH), as well as a decreased number of BrdU+ (red) but not Ki67+ (green) cells as compared to shCtl. cells. (B) Left panel: Live cell-imaging based scratch-wound assay of cells described in (A) at 0, 24, 48 and 72 hours after wounding. Initially, 30 000 cells/well (96-well plate) were seeded, reproducible wounds were scratched using the wound maker tool (Essen bioscience). Scale bars indicate 200 µm, the initial wound is indicated by white dotted lines. Right panel: Quantification of the scratch-wound assay, the wound width [µm] was determined every 3 hours. Shown are mean values ± sdv of n = 8 replicates, p = 3.1E-05 (shCtl.1 vs. sh#3) or 9.6E-07 (shCtl.2 vs. sh#3), ttest. (C) Left and right panel: CD271-responsible regulation of genes associated with formation of cell projections as determined by GSEA of T20/02 cells engineered for stable down-regulation (T20/02k.d.) or over expression (T20/02CD271/NGFR) of CD271. (D) Representation of genes found either up-regulated or down-regulated by knock-down or over expression, respectively. A common set of 20 genes followed a CD271-responsible regulation among them FGF13 and CSPG4. (E) Left panel: Determination of expression levels of potential migration-associated genes CSPG4, FGF13, HMGA2 and AKT3 by qPCR of T20/02 cells either stably transfected with control shRNA (shCtl) or effective CD271-targeting shRNAs (sh#3, sh#4; sh#2, not effective). Relative expression levels are shown as mean values ± sdv of biological triplicates, ***p ≤ 0.001, **p ≤ 0.01, *p ≤ 0.05, ttest. Right panel: Immunofluorescence microscopy for levels of CSPG4, FGF13 and HMGA2 in shCtl. and CD271 knock-down cells (sh#3). DAPI served as nuclear stain, scale bars indicate 50 µm.
