Figure 5.
ABI2 is necessary for hypoxia- and PIM-induced protrusions. (a) DU145 and ABI2-KOA20 cells were stimulated with the indicated doses of EGF in normoxia or hypoxia, and cell area was plotted relative to the pre-stimulated average area. The shaded area represents 95% CI as determined by two-sided Student’s t test (*P < 0.05 between Hyp. 5 nM and Norm. 5 nM and *P < 0.05 between Hyp. 5 nM and Norm. 50 nM; n = 6). (b) DU145 cells were imaged in normoxia and hypoxia in the presence or absence of PIM447 (3 µM). The relative proportion of randomly sampled windows in DU145 or DU145-PIM1-overexpressing cells actively protruding during progressive frames (15-s intervals): DU145 WT (n = 1,637, N = 12), DU145-hPIM1 (n = 1,395, N = 12), and DU145-PIM447 (n = 1,682, N = 11). In hypoxia, DU145 WT (n = 1,268, N = 5), DU145-hPIM1 (n = 1,333, N = 10), and DU145-PIM447 (n = 2,056, N = 10). Error is shown as 95% CI as determined by two-sided Student’s t test (*P < 0.05, *P < 0.05 between normoxic and hypoxic conditions within each cell line). (c) Quantification of unstimulated cell protrusive activity in ABI2-KOA20 (n = 2,116 Nx, N = 9 and n = 1,279 Hx, N = 6), ABI2-KOA20-hPIM1 (n = 1,213 Nx, N = 7 and n = 2,183 Hx, N = 10), and ABI2-KOA20-PIM447 (n = 1,899 Nx, N = 10 and n = 2,133 Hx, N = 10) cells. Error = 95% CI as determined by two-sided Student’s t test (*P < 0.05, *P < 0.05 between normoxic and hypoxic conditions within each cell line). (d) Representative cell traces depicting changes in actin structure over time in DU145 and ABI2-KOA20 cells overexpressing PIM1 or treated with PIM447 under normoxic and hypoxic conditions. Regions representative of changes in actin structure are marked as follows 1 = lamellipodia, 2 = retraction, 3 = transient protrusion. Scale bar = 5 µm for all images.