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Fig. 8
Stiffening TRCs via actin polymerization decreases transmigration in vitro and extravasation in vivo.Transwell migration of TRCs were compared with that of control melanoma cells before or after actin was polymerized by addition of Jasplakinolide. (a) Transmigrated cell nuclei is the percentage of transmigrated cell nuclei (% seeded cells) per view-field. (b) Transmigrated cytoplasmic area is the total transmigrated cytoplasmic area per view-field. For (a) and (b), Mean ± s.e.m.; n = 3 independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001. (c) TRCs and Cont pretreated with or without Jasp were injected into the pericardium of 48 hpf embryos respectively. Representative images were acquired with high resolution confocal microscopy (63x objective), showing penetration of Cont ± Jasp (left panels) or TRCs ± Jasp (right panels) at 12 and 24 hpi respectively. Dashed white lines mark the tumor extravasation areas (various sizes of micrometastases) from vessels to surrounding tissues. + Jasp: tumor cells were pretreated with 100 nM Jasp in culture medium for 12 hr before collecting; - Jasp: tumor cells were pretreated with DMSO at the same concentration to Jasp in culture medium for 12 hr before collecting. Scale bars, 100 µm Insets scale bars, 20 µm. (d) Quantification of extravasated tumor area relative to the total tumor area at different time points: 12, and 24 hpi. Color code: Zebrafish blood vessels are green, and mouse tumor cells are red. TRCs exhibit higher penetration rates than Cont. Mean ± s.e.m.; n >6 fish per group; 3 independent experiments; *p < 0.05.