Figure 4

The zebrafish yolk sac is hypoxic and xenotransplant proliferation requires eIF4E2: (A) TREx-BCBL1-RTA cells were labeled with 1 µM of Image-iT Green Hypoxia Reagent for 30 min prior to washing and labeling with CMTMR dye. Cells were injected into 2 dpf embryos and imaged 1 hour later. Scale bar = 100 µm. (B) TREx-BCBL1-RTA cells were transduced with eIF4E2 shRNA or a non-targeting control lentivirus. Cells were harvested and probed for eIF4E2 and homologue eIF4E1 by Western blotting; (C) TREx-BCBL1-RTA cells or cells transduced as in (B) were seeded at 2.5 × 10⁵ cells/mL and monitored for viability and proliferation by manual counting, using a hemocytometer and trypan blue for the following five days (n = 3 independent transductions; means ± SEM; statistical significance was determined by two-way ANOVA); (D) proliferation of transduced TREx-BCBL1-RTA cells at 3 dpi normalized to the number of cells counted at 1 dpi (n = 3 independent experiments with cells from 20 larvae counted per measurement; means ± SEM; statistical significance was determined by two-way ANOVA compared to the cell counts at 1 dpf; * = p-value < 0.05).