Knockdown of RAGE in THP-1 Macrophages

The RAGE expression in the THP-1 cells has previously been documented [15 (link)]. To obtain a RAGE-THP-1 cell line, the transfection of RAGE shRNA lentiviral particles was used to knock down RAGE gene expression. In brief, the THP-1 cells were resuspended in RPMI-1640 medium with 10% FBS. A total of 1 × 10³ THP-1 cells were transfected with RAGE shRNA lentiviral particles (sc-36374-V, Santa Cruz Biotechnology, Inc., 10410 Finnell Street Dallas, Texas 75220, USA), the nontargeting control sequence (sc-108080), or copGFP control plasmid (sc-108084) in 96-well plates, as recommended by the manufacturer (Santa Cruz Biotechnology, Inc., 10410 Finnell Street Dallas, Texas 75220, USA). Following 1.5 months of puromycin selection, stable cultures of THP-1 cells with RAGE-targeted knockdown were selected and cloned for the induction of macrophages. The efficiency of RAGE-targeted knockdown in the macrophages was examined by western blot analysis.