PEDF maintains stemness and self-renewal of GSCs by activating Notch-Sox2 pathway.

<p>(A) IB analysis of PEDF (in medium) and NICD in X02 cells infected with PEDF-expressing lentiviral or control construct (upper panel) and Ex Vivo cells infected with PEDF-expressing lentiviral or control construct (lower panel). α-tubulin was used as a loading control. (B) IB analysis of PEDF (in medium) and NICD in X01 cells infected with shPEDF2-expressing lentiviral or control construct (upper panel) and CSC2 cells infected with shPEDF2-expressing lentiviral or control construct (lower panel). α-tubulin was used as a loading control. (C) IB analysis of NICD, Sox2, and Nestin in X02-Con, X02-PEDF, or X02-PEDF cells treated with 1 μM of DAPT (γ-secretase inhibitor). (D) Sphere formation assay was performed in X02-Con, X02-PEDF, or X02-PEDF treated with 1 uM of DAPT. The graph represents the average proportion of sphere number. Counted sphere size is greater than 100 μm. All error bars represent mean ± SEM (<i>n</i> = 3). ** <i>p</i> < 0.01. (E) Semiquantitative RT-PCR of PEDF and Sox2 in X02-Con or X02-PEDF (upper panels) and X01-Con or X01-shPEDF2 (lower panels). GAPDH was used as a loading control. (F) Chromatin immunoprecipitation (ChIP assay) was performed in X02-Con or X02-PEDF cells with NICD-specific or control antibodies. The strategy for the ChIP assay is represented in the upper panel. Consensus CBF1 binding site (TGGGAA) located in Sox2 at -1007 and -889. (G) IB analysis of PEDF (in medium), Sox2, Nestin, GFAP, and NICD in X01-Con, X01-shPEDF2, X01-Sox2, or X01-shPEDF2 coinfected with Sox2-expressing lentiviral construct. α-tubulin was used as a loading control. (H) Sphere formation assay was performed in X01-Con, X01-shPEDF2, X01-Sox2, or X01-shPEDF2 coinfected with Sox2-expressing lentiviral construct. The graph represents the average proportion of sphere number. Counted sphere size is greater than 100 μm. All error bars represent mean ± SEM (<i>n</i> = 3). ** <i>p</i> < 0.01.