Figure 1.
Lovastatin inhibits the angiogenesis and MLC phosphorylation of HUVECs in vitro.
(A) HUVECs were pre-treated with various concentrations of lovastatin for 24 h. In vitro analysis of angiogenesis was carried out using capillary tube formation assay (upper panel; scale bar: 1 mm, ×4 objective) and transwell migration assay (lower panel; scale bar: 200 µm, ×20 objective). Note the dose-related decrease in the relative tube area and migrated cells. (B) Quantitative evaluation of the area covered by tubes (tube formation assay) and relative number of migrated cells (transwell assay) from (A). Data represent four independent experiments. (C) HUVECs were treated as in (A) and analyzed for phosphorylated and total MLC levels. β-actin was also probed as an internal control. Signal intensity of pMLC is expressed as percentage of control intensity ratio. n = 4. Data represent mean ± SEM. *, p<0.05, **, p<0.01, ***, p<0.001 compared with unstimulated control.
Figure 2.
Identification of proteins differentially expressed in response to lovastatin treatment.
(A) Representative 2D difference gel electrophoresis (DIGE) images of HUVECs treated with lovastatin (10 µM, 24 h) or vehicle. Among the differentially expressed protein spots, three major spots were selected (arrows). (B) Detailed views of spot 1, 2 and 3 (arrows). (C) The spot 2 was identified as transgelin 2 by ES-Q-TOF-MS analysis followed by database searching, with a Mascot score of 69 and sequence coverage of 16%. The fragment ion spectrum of spot 2 is exhibited.
Figure 3.
Lovastatin increases transgelin 2 expression.
(A) The localization of transgelin 2 in HUVECs. Confocal microscopic examination showed the association (yellow) of transgelin 2 (green) with actin filaments (red). (B) The localization of transgelin 2 (green) in HUVECs after lovastatin treatment (10 µM, 24 h) was visualized under confocal microscope (40× magnification). The negative control shows a confocal image without primary antibodies. Hoechst was used for nuclei staining (blue). Scale bar: 50 µm. (C) Western blot analysis of HUVECs treated with the indicated doses of lovastatin for 24 h. Bands were quantitated by densitometry and normalized against β-actin (n = 3). (D) Western blot analysis of HUVECs treated with lovastatin (10 µM) for the indicated times. Bands were quantitated by densitometry and normalized against β-actin (n = 4). Each data point represents mean ± SEM. *, p<0.05; **, p<0.01 compared with unstimulated control. (E) Transgelin 2 expression tended to be increased by the ROCK inhibitor Y-27632 (20 µM, 24 h) and the actin polymerization inhibitor cytochalasin B (cytoB, 5 µM, 24 h) compared to control (ctrl).
Figure 4.
Effects of transgelin 2 knock-down on HUVECs migration and tube formation.
(A) Immunoblotting verified the degree of transgelin 2 (TAGLN2) depletion by siRNA. β-actin was employed as loading control. (B) After 24 h of siRNA transfection, cells were treated with lovastatin (10 µM) or vehicle for another 24 h and analyzed for cell migration (scale bar: 200 µm, ×20 objective). Data are evaluated for the average number of migrated cells relative to basal conditions with control siRNA-transfection (n = 6). (C) Cells treated as in (B) were analyzed for tube formation (scale bar: 1 mm, ×4 objective). Data are evaluated for the area covered by tubes relative to basal conditions with control siRNA-transfection (n = 5). Data represent mean ± SEM. *, p<0.05; ***, p<0.001 versus control siRNA-transfected untreated cells. #, p<0.05.
Figure 5.
Transgelin 2 regulates HUVECs pMLC levels and Rho GTPases activation.
HUVECs were transfected with transgelin 2 siRNA or a scrambled control, and followed by lovastatin (10 µM) treatment as described in Figure 4. (A) Western blot analysis of pMLC and tMLC in HUVECs. β-actin is employed as loading control. pMLC levels were quantitated by densitometry and normalized against β-actin (n = 3). Each data point represents mean ± SEM. p<0.05; **, p<0.01 versus control siRNA-transfected untreated cells; #, p<0.05. (B) Cell lysates were immunoprecipitated with GST-RBD conjugated beads, and active Rho GTPase was detected by immunoblotting. Total cell lysates were subjected to immunoblotting to determine the expression level of total Rho. Experiments were repeated at least three times.