Sc 36374 5

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.

Primary keratinocytes were infected with RAGE sh lentiviral particals (sc-36374-v) and control sh viral particals (sc-108080), following the protocol conditions (Santa Cruz Biotechnology). Positive clones were selected by puromycine selection. Stimulation of shRAGE and sh control expressing cells with S100A8/A9 protein was performed when the cells have reached an appropriate density. For determination of the effect of RAGE knockdown on cellular proliferation with a following S100A8/A9 stimulation, BrdU proliferation assay was performed.