eIF4E2 is required for MDA-MB-231 cell migration, invasion and spheroid formation in hypoxia. (a) Western blot of eIF4E2 protein levels in control (Ctrl) cells stably expressing a non-targeting shRNA or in cells stably expressing one of two shRNAs targeting eIF4E2 mRNA: Knockdown (KD) 1 and KD2. Two clones of each stable cell line were generated: KD1.1, KD1.2, KD2.1 and KD2.2. GAPDH used as a loading control. (b) Control and eIF4E2 KD cells exposed to normoxia (21% O2) or hypoxia (1% O2) for 24 h followed by wound generation. Representative images at 0 h (T0) and 16 h (T16) after wound generation. (c, d) Transwell migration (c) and invasion (d) assays of control and eIF4E2 KD cells exposed to normoxia or hypoxia for 24 h. Representative images of transwell inserts 16 h after seeding and stained with crystal violet. (e) Light micrographs of spheroids composed of control cells or eIF4E2-depleted cells (KD1.2 and 2.1). Data are presented relative to normoxic Ctrl as mean ±s.e.m., n ≥3, ***P<0.001, using a one-way ANOVA followed by Tukey’s HSD test. Scale bar, 100 μm.