Fig. S1.

Hyperglycemia induces HSP22 upregulation and endothelial activationin vitro. Human umbilical vein endothelial cells (HUVECs) were cultured in endothelial basal medium (EBM) containing normal glucose (NG, 5 mM D-Glucose), osmotic control (OC, 30 mM D-mannitol), or high glucose (HG, 30 mM D-Glucose) for 24 h. A) Cell viability was evaluated in cells in 96-well plates by a Cell Counting Kit-8 (CCK8) after treatment with NG, OC or HG for 24 h. n = 3. B) HUVECs were cultured in EBM containing NG, OC or HG for 24 h, and the cell media were collected for the measurement of lactate dehydrogenase (LDH) release as an indicator of cytotoxicity. n = 3. C) HUVECs were cultured in EBM containing NG, OC or HG for 24 h, and the adhesion of nonstimulated primary human peripheral mononuclear cells (PBMCs) to stimulated HUVECs was quantified. n = 3. D) mRNA expression of ICAM-1 and VCAM-1 was determined by RT-PCR. n = 3. E) Endothelial cell activation-related cytokines were examined by RT-PCR. n = 3. F) Representative immunofluorescence staining images of HSP22 (green), the endothelial cell marker CD31 (red), and nuclei (blue) (original magnification ×63). G) Quantification of the fluorescence intensity of HSP22 and CD31 in HUVECs. n = 3. H) mRNA expression of HSP22 was determined by RT-PCR. n = 3. I) HSP22 protein expression was determined by Western blotting. J) Quantification of HSP22 protein expression. GAPDH was used as a loading control. n = 3. ^*P < 0.05 vs. NG; ^#P < 0.05 vs. OC.