Oxldl

Monocytes isolated from PBMCs of healthy individuals using the Robosep^™ monocyte isolation kit (StemCell, MA, USA) were cultured as 500,000 cells per 250 μL X-VIVO 15 (Lonza, Basel, Switzerland) media supplemented with 10% FBS and 1% penicillin-streptomycin. Anti-LOX-1 antibody (50 μg/mL) or Control IgG antibody (Clone: NIP228) were used to pre-treat monocytes for 30 min at 37˚ C. Cells were then treated with 30 μg/mL oxLDL (Cell biolabs, CA, USA) for 3 h and then stimulated with DNA-immune complexes (1 μg/mL CG50 + 20 μg/mL E11 IgG antibody) for 24 h. Supernatants were then collected, diluted at 1:6 ratio and added to 7-Plex Human Proinflammatory cytokine plates (MesoScale Diagnostics, MD, USA) to detect various cytokines.

HUVECs were purchased from American Type Culture Collection (ATCC) and cultured in recommended conditions. Cells of the same batch were used in all experiments. SiRNA and pCMV6 vectors of IκBβ and wide/mutant lnc-MKI67IP-3 were obtained from GenePharma (Shanghai, China) or Origene (Rockville, USA). The mimic and inhibitor of let-7e and corresponding negative controls (NC) were bought from GenePharma. Vitamin C was purchased from Sigma Chemical Co. (St. Louis, USA). Human ox-LDL was purchased from Yiyuan Biotechnologies (Guangzhou, China) and it was prepared as previously described43 (link). Oxidation of ox-LDL was confirmed by thiobarbituric acid-reactive substances (TBARS) assay according to the manufacturer’s instructions (CellBiolabs, USA). HUVECs were treated with ox-LDL (50 υg/ml) or Vitamin C and then were harvested at 12, 24 and 48 hours after treatment. The mimic/inhibitor/NC of let-7e, siRNA or different vectors, respectively, were transfected into HUVECs using lipofectamine RNAIMAX or 3000 reagent (Invitrogen, Carlsbad, USA). HUVECs were harvested 24 hours after transfection for subsequent experiments.