Hypoxia increases tumor cell invasion and protrusive activity. (a) Representative images of Boyden chambers invasion assay in normoxia (Nx) or hypoxia (Hx; 1% O₂), with accompanying quantification of cell number (n = 3). (b) Quantification of DU145 cell area following stimulation with EGF (5 nM) and IGF (5 nM; n > 10 cells/condition). Values are relative to cell area prior to stimulation. (c) Representative heatmap of basal (unstimulated) protrusive velocity of DU145 cells in normoxia (n = 1,637, N = 5) and hypoxia (n = 1,286, N = 12). (d) Quantification of relative protrusion activity in normoxia and hypoxia for DU145 (n = 14) and MCF7 (n > 9) cells. Error is shown as 95% CI by two-sided Student’s t test (*P < 0.05, **P < 0.01). (e) Representative images of 3D invasion assays using PC3-LN4 and PC3-LN4 PIM1-KO cells in normoxia and hypoxia. Invading cells are marked in red, and the number of invading cells is indicated in the upper left corner. Scale bar = 200 µm for all images. (f and g) Quantification of the total number of invading cells and (g) average invasion distance from the core of PC3-LN4 (n = 2,864 Nx and n = 2,079 Hx) and PC3-LN4 PIM1-KO (n = 255 Nx and n = 143 Hx) cells. N = 3 biological replicates for all conditions. Error is shown as 95% CI by two-sided Student’s t test (*P < 0.05).