FIG 2.

TIE2 nuclear translocation is caveolin-1 dependent. (A) U251.Tie2 cells were IR treated (¹³⁷Cs; 10 Gy) for the indicated time periods, and nuclear and cytoplasmic protein extraction was performed by subcellular fractionation, as previously described (3). Proteins were analyzed by Western blotting, as reported previously (3), to assess TIE2 and caveolin-1 (CAV1) expression levels. LaminB and tubulin expression levels were used as nuclear and cytoplasmic protein loading controls, respectively. γH2AX expression levels were used as an indicator of DNA damage after IR. (B) U251.Tie2 cells were serum starved for 24 h in serum-free medium and then incubated in fresh serum-free medium containing the caveolin-1 inhibitor filipin (5 μg/ml; Sigma-Aldrich); 60 min later, Ang1 (400 ng/ml; R&D Systems) was added for 30 min. Proteins were extracted after nuclear and cytoplasmic fractionation, as previously reported (3), and levels of TIE2 and caveolin-1 expression were assessed by Western blotting. DMSO was used as the vehicle (Veh.). LaminB and tubulin expression levels were used as nuclear and cytoplasmic protein loading controls, respectively. (C) siRNA against caveolin-1 was transfected into U251.Tie2 cells, and 48 h later, cytoplasmic and nuclear TIE2 and caveolin-1 were analyzed by Western blotting after Ang1 exposure (400 ng/ml; 30 min). Bovine serum albumin (0.1% in phosphate-buffered saline) was used as the vehicle/control in Ang1(−)-treated samples. siRNAs were transfected into cells using the INTERFERin transfection reagent (PolyPlus Transfection) at concentrations of 10 nM; after 48 h, subcellular fractions of nuclear and cytoplasmic proteins were analyzed by Western blotting. Nontargeted siRNA (siCon) was used as the control. (D) Western blot analysis with cytoplasmic and nuclear extract from U251.Tie2 cells after transfection of siRNA against Tie2 (10 nM) and Ang1 stimulus, as explained for panel C. The antibodies and working dilutions are listed in Table S3. The siRNA sequences used are listed in Table S1. (E) U251.Tie2 cells were plated in a 96-well plate at subconfluent density and treated with Tie2-13 (5 μM) and filipin (2 μg/ml) (36), alone or in combination; 24 h later, they were irradiated (15 Gy) and kept in an incubator for an additional 48 h at 37°C. Cell viability was assessed using the CellTitre-Blue cell viability assay (Promega) according to the manufacturer's instructions. Data represent means ± standard deviations (SD) from three independent experiments. P values were calculated using Welch's modified t test; ***, P ≤ 0.001. (F) GSC-20 cells were treated as described for panel B with Tie2-13 (5 μM) and filipin (1 μg/ml), alone or in combination for 1 h, and then irradiated (20 Gy) and kept in an incubator for an additional 72 h at 37°C. Data represent means ± SD from three independent experiments. P values were calculated using Welch's modified t test; ***, P ≤ 0.001.