VEGF expression is positively regulated by CaMKII in human OS.

<p>143B cells were transduced with lentiviruses expressing either scrambled (shCtrl) or α-CaMKII- targeting shRNAs (shCaMKIIα) or treated with the CaMKII inhibitor KN-93. HOS cells were transduced with retroviruses expressing either GFP (GFP-Ctrl) or CaMKIIα (GFP- CaMKIIα). <b>A.</b> Real-time PCR was performed using primers specific for VEGF or β-Actin. Values were obtained from three separate experiments each repeated in triplicate and represent the mean ±S.D. *p<0.01. <b>B.</b> α-CaMKII-inhibited 143B (shCaMKIIα or KN-93) and α-CaMKII overexpressing HOS (GFP-CaMKIIα) cells were seeded at 1 x 10⁵ cells per well of a 6-well plate. Twenty-four hours later, aliquots of supernatant were examined for human VEGF by ELISA. Values were obtained from three separate experiments each repeated in triplicate and represent the mean ±S.D. *p<0.01. <b>C.</b> α-CaMKII inhibited 143B (shCaMKIIα or KN-93) and α- CaMKII overexpressing HOS (GFP-CaMKIIα) cells were seeded at 1 x 10⁵ cells per well of a 6- well plate. Twenty-four hours later, aliquots of conditioned media were removed and added to 12-well plates seeded with 1 x 10⁵ HUVEC cells. Endothelial cell tube-like formation was measured at 12 h. Negative controls of non-conditioned culture media (DMEM + 10% FBS) and conditioned media with VEGF immunoprecipitated out (VEGF IP) were also included. Representative photomicrographs were taken at 50x magnification from 3 independent experiments, each repeated in triplicate. Scale bar = 50 μm <b>D.</b> Capillary tube-like length was quantified using the ImageJ software. Values were obtained from three separate experiments each repeated in triplicate and represent the mean ±S.D. *p<0.01.