Fig. 3: TRF2 is required for productive 1D and 3D migration.
A Schematic representation of cells on fibronectin coated plates for random migration analysis (2D migration). B Schematic representation of cells on micropatterned lines, showing a polarized migratory morphology for 1D migration analysis. C Box-and-whisker plots (10-90 percentile) representing velocity, effective length, and persistence parameters of MDA-MB-231 cells stably depleted for TRF2 (shTRF2 N1) random migrating on the plate. D Box-and-whisker plots (10-90 percentile) representing velocity, effective length, and persistence parameters of MDA-MB-231 cells stably overexpressing TRF2 random migrating on the plate. E Box-and-whisker plots (10–90 percentile) representing velocity, effective length, and persistence parameters of MDA-MB-231 cells stably depleted for TRF2 (shTRF2 N1) migrating on micropatterned lines. F Box-and-whisker plots (10–90 percentile) representing velocity, effective length and persistence parameters of MDA-MB-231 cells stably overexpressing TRF2 migrating on micropatterned lines. G For 3D spheroid invasion assay, MDA-MB-231 stable cell lines were plated in a 96 well plate with a specialized ECMR to drive spheroid formation of the cells. After 3 days (day 0) spheroids were embedded in Invasion MatrixR and 3D invasion was visualized microscopically at indicated time points. Left panel: invasive activity into ECM was expressed as total spheroid area. Data are the mean ± SD (n = 3 independent experiments). Right panel: phase contrast images of representative spheroids for the indicated experimental conditions at day 4 are shown. Dashed circle indicates the size of spheroids at day 0. Scale bars, 100 μm. H MDA-MB-231 cells stable overexpressing TRF2 WT (pTRF2) or TRF2_ΔB (pTRF2_ΔB) deletion mutant and their control (pBabe) were subjected to 3D spheroid invasion assay. Left panel: invasive activity into ECM was expressed as total spheroid area. Data are the mean ± SD (n = 3 independent experiments). Right panel: phase contrast images of representative spheroids at day 4 are shown. Dashed circle indicates the size of spheroids at day 0. Scale bars, 100 μm. For (C, D and E, F), two independent experiments were performed. At least 600 cells were analyzed. Kolmogorov-Smirnov test was used to calculate statistical significance. For (G, H), Two-tailed t student test was used to calculate statistical significance. *P < 0.05, **P < 0.01, ***P < 0.001.
