Fig 2: The effect of Oba and MIF inhibitor ISO-1 on CPPS. (A) The protein level of MIF in (A) serum and (B) prostate of EAP mice. N = 4/group. Unpaired t-test. (C) The prostatodynia was assayed 7 days after MIF inhibitor application (Sham + vehicle group vs. EAP + vehicle group, ***p < 0.001 at 1 g and 4 g; EAP + vehicle group vs. EAP + ISO-1 [M] group, #p < 0.05 at 1 g). (D) The effect of Oba [M] on prostatodynia in the presence or absent of ISO-1 [M] (EAP + vehicle vs. EAP + ISO-1 [M], #p < 0.05 at 1 g; EAP + vehicle vs. EAP + Oba [M], *p < 0.05 at 1 g, **p < 0.01 at 4 g). N = 8–10/group. Two-way ANOVA followed by Bonferroni multiple comparisons test. All the values are shown as means ± SEM. Significant difference was considered when P < 0.05.

Fig 3: Radiation improved tumor hypoxia and impeded HIF-1α-dependent MIF secretion by NSCLC. a Representative microphotographs of immunofluorescence staining showing expression of CD34(red) and α-SMA (green) in shNC or shMIF BM mouse model with or without 10 Gy radiation therapy (n = 3). b. IHC staining of HIF-1α in brain metastatic tissue of BM mouse model. c. Correlation analysis of MIF and HIF-1α in NSCLC patients in R2 database. d. Dose–response curves of KC7F2 in Lewis cell. e. Western blotting for the level of HIF-1α and MIF in Lewis cell under 1% O2 with 0 μM, 10μ, 20 μM, 30 μM, 60 μM KC7F2. f. Western blotting for the expression of HIF-1α and MIF in Lewis cell under 1% O2 after 3 h, 6 h, 12 h, 24 h, 48 h. g. Western blotting for the expression of HIF-1α and MIF in Lewis cell under 1% O2 without KC7F2 and with 30 μM KC7F2 after 3 h, 6 h, 12 h, 24 h, 48 h. h. Change of MIF concentration in Lewis cell’s supernatant under 21% or 1% O2 with or without KC7F2 (n = 3). i. Binding ability of HIF-1α and MIF promoter in EMSA assay. j. Western blotting for the expression of iNOS, CD86, Arg-1 and YM-1 in shNC or shCD74 BV2 cell of the co-culture system. 30 μM KC7F2 was added to the culture medium of Lewis cell in the co-culture system. k. Representative microphotographs of immunofluorescence staining showing expression of Arg-1 and CD86 in shNC or shCD74 BV2 cell of the co-culture system. 30 μM KC7F2 was added to the culture medium of Lewis cell in the co-culture system

Fig 4: MIF inhibition enhanced radiosensitivity for brain metastasis via synergistically promoting microglia M1 polarization in vivo a. The schema for animal studies. b. Representative bioluminescent imaging of BM model assay with shNC and shMIF Luc-Lewis cells carotid artery injected in C57BL/6 mice with or without 10 Gy radiotherapy(n = 10). c. HE staining of BM mice model assay. d. Survival analysis for BM mice model (n = 5). e. Body weight measurement for BM mice model (n = 5). f. Western blotting for the expression of iNOS and Arg-1 in brain metastasis of BM model. g. Immunofluorescence staining of brain metastasis tissue of BM mice model. h. At 7 days after radiotherapy, the ratio of CD86 positive and CD206 positive BV-2 cell was analyzed by flow cytometry and the data was processed with FlowJo (version 10.0) program (left). The mean fluorescence intensity of CD86 and CD206 in BV-2 cell was analyzed by flow cytometry and the data was processed with FlowJo (version 10.0) program(right, n = 3). i. At 14 days after radiotherapy, the ratio of CD86 positive and CD206 positive BV-2 cell was analyzed by flow cytometry and the data was processed with FlowJo (version 10.0) program (left). The mean fluorescence intensity of CD86 and CD206 in BV-2 cell was analyzed by flow cytometry and the data was processed with FlowJo(version 10.0) program(right, n = 3)

Fig 5: Blocking the MIF-CD74 interaction between NSCLC and microglia promoted microglia M1 polarization a. Co-culture sketch map of CD74-shRNA lentivirus transduced BV2 cells and MIF-shRNA lentivirus transduced Lewis cells. After 16-Gy radiation, BV2 cell were immediately co cultured with Lewis cells for 24 h. b. Total RNA of BV2 cell was extracted after 24 h co-culture, and mRNA expressions of iNOS, Arg-1, CD86 and YM-1 were analyzed by real-time PCR (n = 3). c. Western blotting for the expression of iNOS, CD86, Arg-1 and YM-1 in BV2 cell after 24 h co-culture. d. Representative fluorescent images showing different phagocytosis of microspheres (green) with phalloidin (red) in BV-2 after 24 h co-culture. e. Representative microphotographs of immunofluorescence staining showing expression of Arg-1(left) and CD86(right) in BV-2 cell after 24 h co-culture. f. The ratio of CD86 positive and CD206 positive BV-2 cell was analyzed by flow cytometry and the data was processed with FlowJo (version 10.0) program (n = 3). g. The mean fluorescence intensity of CD86 and CD206 in BV-2 cell was analyzed by flow cytometry and the data was processed with FlowJo (version 10.0) program (n = 3)