USP28 is involved in hypoxia and HIF-1–dependent capillary formation. (A) HMEC-1 cells were transfected with vectors expressing USP28 shRNA1 or USP28 shRNA3 or control shRNA (shCtr). Cells were seeded onto Matrigel-coated wells for 2 hours and then exposed to hypoxia (H) or normoxia (N) for 6 hours. The number of tubules from each well was counted using ImageJ Version 1.457 software. Data represent the number of tubules relative to the control, which was set to 100%. *Significant difference between the shRNA control vector versus USP28 shRNA vectors under normoxia. **Significant difference between the shRNA control vector versus USP28 shRNA vectors under hypoxia. (B) Photographs from a representative in vitro angiogenesis experiment. (C) HMEC-1 cells were transfected with vectors for control shRNA (shCtr) or shRNA against HIF-1α (shHIF-1α) alone or in combination with a vector expressing USP28. Cells were plated onto Matrigel-coated wells as described in panel A. *Significant difference between the control vector versus USP28 and shRNA under normoxia; **significant difference between the control vector versus USP28 and shRNA under hypoxia. (D) Photographs from a representative in vitro angiogenesis experiment.