Fig 2: CXCL1 mediated the proliferation of C6 cells induced by exogenous GDNF.(A) CXCL1 promoter activity increased significantly after GDNF treatment by double luciferase reporter gene activity. pGL 4.20 was an empty carrier. pRL-TK was internal reference. (B) The mRNA level of CXCL1 was analyzed by q-PCR. (C) Cell supernatants were collected and the secretion level of CXCL1 was detected by ELISA. (D) CCK-8 was used to detect the cell activity of each group. (*P<0.05, **P<0.01,***P<0.001).

Fig 3: NF-κB mediated the effect of GDNF on CXCL1 and proliferation in rat C6 glioma cells.(A) Western blot showed that NF-κB mediated the effect of exogenous GDNF on CXCL1. (B) The statistical results of (A). (C) NF-κB mediated GDNF enhanced the activity of CXCL1 gene promoter region by double luciferase reporter gene activity. pGL 4.20 was an empty carrier. pRL-TK was internal reference. (D) CCK-8 results showed NF-κB mediated the proliferation of C6 cells by exogenous GDNF. (*P<0.05, **P<0.01, ***P<0.001).

Fig 4: GDNF promoted NF-?B nuclear transformation and the interaction CXCEL with NF-?B.(A) Transcription factors that regulate the gene CXCL1. Qiagen online software was used to analyze the TF that initiated CXCL1 transcription and its corresponding BS. (B) Exogenous GDNF promoted the interaction CXCEL with NF-?B by ChIP-qPCR assay. (C) Western blot showed exogenous GDNF promoted NF-?B nuclear transformation. The C6 cells were treated with exogenous GDNF for 48h, and nuclear protein were extracted to determine NF-?B (p65) levels by Western blot. Histone 3 was used as internal references. (D) The statistical results of (C). (E) NF-?B nuclear transformation was analyzed by Immunofluorescence. The C6 cells were treated with exogenous GDNF for 48h and fixed with 4% paraformaldehyde (PFA) for Immunofluorescence. Bar = 200 µm. (*P<0.05,**P<0.01, ***P<0.001).

Fig 5: The expression level of CXCL1 in C6 cells and GBM.(A) The correlation between CXCL1 and the overall survival of GBM patients on GEPIA. (B) The expression of CXCL1 in GBM tissues compared with related normal tissues. (C) Identification of AST. AST transmitted to the third generation were stained with anti-GFAP immunofluorescence. The cytoplasm of positive cells was stained red, and the nucleus of DAPI stained blue. Bar = 200 μm. (D) Western blot showed that CXCL1 protein expression level in C6 cells and AST. (E) The statistical results of (D). (F) ELISA showed that CXCL1 supernatant content in C6 cells and AST. (**P<0.01, ***P<0.001).