Application: WB
Species: Human
Sample: HCAECs
Figure 2
Orai1–3 protein and IGFBP3 expression levels and SOCE activity are significantly increased in HCAECs cultured in HG for 7 days and coronary artery endothelial cells of streptozotocin-induced diabetic mice with no change in STIM1 expression levels; increased Orais promotes proliferation of HCAECs and decreased IGFBP3 reduces proliferation of HCAECs. (A, C) Representative traces and (B, D) summary data showing the maximum increase in SOCE in HG-cultured HCAECs. After treatment of HCAECs with either 2 µM TG or 100 µM ATP for 10 min, application of 2 mM Ca2+ induces significant increase in [Ca2+]i through SOCE. (E, G, I, K, A') Representative western blot images and (F, H, J, L, B'–E') summary data showing Orai1, Orai2, Orai3, STIM1 and IGFBP3 expression levels in HCAECs cultured in NG or HG medium. (M, O, Q, F') Representative images of coronary artery endothelium immunostaining (brown; eg, red arrowheads) for protein expression levels of Orai1–3 and IGFBP3 in a mouse model of DM and control mice. Magnification, ×400. Scale bar, 50 µm, applies to all panels. (N, P, R, G') Quantification of Orai1–3 and IGFBP3 protein expression levels in the coronary artery endothelium of a mouse model of DM and control mice. (S) Representative western blot images and (T) summary data showing PCNA protein expression levels in HCAECs cultured in HG or NG medium for 7 days in the presence or absence of the SOCE agonist ATP (100 µM) or the SOCE inhibitor BTP2 (10 µM). (H') Representative western blot images and (I') summary data showing IGFBP3 siRNA knockdown of PCNA protein level. (U) Viability of HCAECs cultured in NG or HG medium for 7 days in the presence or absence of ATP (100 µM) or BTP2 (10 µM) as detected using the CCK-8 assay. (J') Results of CCK-8 assay examining HCAEC viability after cells were cultured in either NG or HG medium for 7 days and then transfected with either IGFBP3-specific siRNA or scrambled siRNA. β-tubulin or GAPDH was used as loading controls. Values represent mean±SEM (n=4–21 samples). *P<0.05, **P<0.01, ***P<0.001 compared with NG-cultured cells or control groups. BTP2, N-[4-[3,5-Bis(trifluoromethyl)-1H-pyrazol-1-yl]phenyl]-4-methyl-1,2,3-thiadiazole-5-carboxamide; CCK-8, Cell Count Kit-8; DM, diabetes mellitus; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HCAECs, human coronary artery endothelial cells; HG, high glucose; IGFBP3, insulin-like growth factor binding protein 3; IOD, integrated optical density; NG, normal glucose; PCNA, proliferating cell nuclear antigen; siRNA, small interfering RNA; SOCE, store-operated Ca2+ entry; STIM1, stromal interaction molecule 1; TG, thapsigargin.
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