Fig. 5

Notch1 is a potential NKAP target gene associated with the TME of glioma. a, Gene expression profile of control and NKAP knockdown U87 cells. Red: High level expression; Green: Low level expression. b, mRNA expression levels of Notch family and Notch signaling pathway genes were determined by qRT-PCR. Data are presented as the mean ± s.d. of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.005 vs. Con. c, The protein levels of NICD1 and HES1 were analyzed by western blot in the NKAP knockdown cell lines. GAPDH serves as a loading control. d, The full length human Notch1 promoter (− 2000 bp to + 0 bp) was cloned into the luciferase reporter vector. Notch1 transcription activity was examined after down-regulating the expression of NKAP in U87 and U251 cell lines. e, The lentiviruses carried GFP gene, so the NKAP knockdown cells showed green fluorescence. Notch1 (red) expression was significantly decreased in the NKAP knockdown U87 cells (green). f, ChIP analysis was performed by using antibody against NKAP with primers targeted to the promoter region of Notch1. Isotype-matched IgG was used as a negative control. PRL30-matched H3 was used as a positive control. g, Notch1 and SDF1 expressions were significantly decreased in the NKAP knockdown glioma tissues. h, Downregulation of NKAP reduced the proportion of CD206^high macrophages in the mouse brain glioma tissues. Scale bar = 20 μm. Data are presented as the mean ± s.d. of three independent experiments. ***P < 0.001, **P < 0.01