Fig. 4.

PDGF-AB induces Endo-MT through NF-kB-mediated Snail expression in ECs. a Human brain microvacular ECs were treated with glioma-CM. RNA was isolated and subjected to RNA-seq analysis. Left, heat map for expression of EMT-related transcriptional factors. Right, fold change of these transcriptional factors (n = 3, mean ± SEM). b, c ECs were treated with 100 ng/ml PDGF-AA, PDGF-AB, and PDGF-BB. b Cell lysates were immunoblotted. c RNA was isolated and analyzed by RT-PCR. Results were normalized with GAPDH levels (n = 3, mean ± SEM). d ECs were transfected with siRNAs targeting Snail or control scrambled sequence, and treated with PDGF-AB. Cell lysates were immunoblotted. e ECs were transfected with siRNAs targeting Erg-1, NF-κB, or control scrambled sequence, and treated with PDGF-AB. Cell lysates were immunoblotted. f ECs were treated with PDGF-AB for 2 h. Cells were analyzed by immunofluorescence. g, h ECs were treated with PDGF-AB or control medium for 8 h. Nuclear extracts were immunoprecipitated with anti-NF-κB antibody or IgG, and subjected to ChIP analysis with primers #1 and #2. g DNA was resolved by agarose electrophoresis, and imaged. Shown are representative results with primer #2. The arrow indicates the amplified DNA in Snail promoter. h Quantitative PCR analysis (n = 3, mean ± SEM). i ECs were transfected with siRNAs targeting Snail or control scrambled sequence, 4 days after treatment with Ki8751, followed by cell viability analysis (n = 3, mean ± SD)