Fig 2: MIF enhances the EMT and VM formation in GBM cells(A/B) Western Blots were performed to analyze the expression level of mesenchymal markers (N-cadherin, Vimentin) in U87 and U251 cells treated with ectogenic rhMIF (50ng/ml, 100ng/ml) for 24h or transfected with siMIF. Quantitative relative analyses were calculated following Imagej analysis. The mRNA levels of EMT markers were also detected by Q-PCR. The data shown are the mean ± SD of independent experiments, n = 3. (C) Representative images of immunofluorescence showed the expression levels of E-cadherin in U87and U251cells treated with ectogenic rhMIF (100ng/ml) and transfected with siMIF compared to control (magnification 200X). (D) VM tube formation assay was performed with U87 cells as indicated. Representative images were shown at magnification 400X. Numbers of tubes per file are shown. The data shown are the mean ± SD of independent experiments, n = 3. NC, negative control. *P < 0.05, **P < 0.01, Student's 2-tailed t test or one-way ANOVA.

Fig 3: VM formation is correlated with the high co-expression of MIF and CXCR4 in glioma tissues(A) Representative images showed the VMs (black arrow) and vessels (green arrow) in glioma speicimens. There were red blood cells in the center of the VMs and vessels (magnification: 400×). Basement membrane-like structures were stained by PAS (pink) and endothelial cells were stained by CD34 (brown). Correlation of glioma grades and VM, MIF and CXCR4 high co-expressed glioma specimens and VM were analyzed by Fisher's exact test. (B) Immunohistochemistry was performed to detect HIF1a, MIF and CXCR4 in serial sections of GBM (the top row 100X, the bottom row 200X, a large view of the tumor section was to indicate the VM). (C) Kaplan–Meier analysis of survival for 25 glioma patients stratified by MIF, CXCR4 expression or VM-positive. Low = patients with immunostaining score less than 9. High = patients with immunostaining score equal to 9. A Wilcoxon rank sum test was used to compare survival curves. *P < 0.05; **P < 0.01.

Fig 4: The expression of MIF and CXCR4 is positively correlated with HIF1a in glioma tissues(A) Representative images of normal brain tissues and different grades of glioma tissues showed the immunostaining of HIF1a, MIF and CXCR4 (magnification, 200×). (B) Statistic data of the immunostaining scores were analyzed showing the correlation of the expression of proteins with the grade of gliomas. (C) Correlation of HIF1a expression with MIF and CXCR4 was analyzed in 25 glioma specimens. The linear regression coefficient and statistical significance was indicated the positive correlation of HIF1a with MIF and CXCR4. (D) Double immunofluorescence staining were performed and merged to show co-localization (magnification: 200×). A large view of the co-localization was shown. HIF1a was labeled with red fluorescence. MIF and CXCR4 were labeled with green fluorescence. *P < 0.05, **P < 0.01, ***P< 0.0001, one-way ANOVA or linear regression.

Fig 5: Hypoxia induces VM formation through the MIF-CXCR4-AKT-EMT pathway in glioblastoma cells(A) EMT markers, P-AKT and total AKT expression levels were detected by western blot in U87 cells treated with rhMIF and AMD3100 (a CXCR4 antagonist, 200 nM) or LY294002 (a PI3K inhibitor, 10 µg/ml). Western blots were also used to analyze the expression levels of proteins in U87 cells transfected with siMIF and treated with AMD3100, LY294002, or ISO-1(a MIF antagonist, 20 ug/ml) under hypoxia. (B/C) Q-PCR and immunofluorescence were used to detect the mRNA and protein levels of E-cadherin in U87 cells with different treatments as indicated. The data shown are the mean ± SD of independent experiments, n = 3. (D) U87 cells with the same treatments as (c/d) were analyzed by the VM tube formation assay. Representative images are shown at 400X magnification. Numbers of tubes per file are shown. The data shown are the mean ± SD of independent experiments, n = 3. NC, negative control; C, control. *P < 0.05, **P<0.01, one-way ANOVA.