Figure 4.

Hypoxia and PIM1 increase WRC levels and activation. (a) DU145 or ABI2-KO_A20 cells stably expressing vector or PIM1 were cultured in normoxia or hypoxia (1.0% O₂) for 16 h, and Western blotting was used to assess protein levels. (b) 293T cells were transfected with HA-ABI2 and GFP-WAVE2 and cultured in normoxia or hypoxia for 4 h. ABI2 was immunoprecipitated using anti-HA beads, and WAVE2 binding was assessed by Western blotting for GFP. (c) PC3-dox PIM1 cells were transfected with GFP-WAVE2 and RFP-ARP3. Then, cells were placed in normoxia or hypoxia and treated with dox (100 ng/ml) for 4 h in the presence of DMSO or PIM447 (3 µM). 5 min prior to harvest, all samples were stimulated with EGF (50 nM). WAVE2 was immunoprecipitated using the GFP tag, and ARP3 binding was assessed by Western blotting for RFP. (d) WT or TKO MEFs stably expressing GFP-Lifeact and RFP-ARP3 were starved for 4 h prior to stimulation with PDGF for 15 min. Cells were fixed for imaging, and ARP3 intensity was measured from the edge of cellular protrusions toward the center of the cell body (red dotted line). (e) Quantification of ARP3 intensity at given increments of distance from the leading edge (n > 15 cells/group). * P < 0.05 using two-sided Student’s t test, error = SEM. Scale bar = 20 µm for widefield images and 4 µm for insets. Source data are available for this figure: SourceData F4.