Fig 1: IL-6 downregulates protein tyrosine phosphatase, receptor type O (PTPRO) in hepatocellular carcinoma (HCC) monocytes by upregulating miR-25–3 p via STAT3/c-MYC. (A, B) Heatmap and volcano plot indicating significantly expressed miRNA within monocytes in patients with HCC compared with the healthy controls. (C) Anti-CD14 magnetic beads were used to isolate human monocytes from 165 patients with HCC and 155 healthy controls. Transcription of miR-25–3 p was detected using real-time PCR and compared between the two groups. **p<0.01, Student’s t-test. (D, E) The linear correlations between expression of miR-25–3 p and PTPRO or PD-L1 were analyzed in HCC monocytes. (F) The potential binding site and mutation in the 3′ untranslated region (UTR) are indicated in the schematic figure. A luciferase reporter gene assay was carried out to test the promoter activity of PTPRO regulated by miR-25–3 p on the 3′UTR of PTPRO. (G, H) Linear correlations between serum IL-6 and monocyte PTPRO, and serum IL-6 and monocyte miR-25–3 p were analyzed in patients with HCC. (I) THP-1–derived and U937-derived macrophages were treated as indicated, and expression of miR-25–3 p was determined by real-time PCR. **p<0.01, Student’s t-test, compared with control group. (J) Cell signaling was analyzed by western blotting. Each experiment was performed in triplicate. Data are presented as the mean±SEM and were analyzed with Student’s t-test (**p<0.01).
Fig 2: IL-6 activates both the uptake and utilization of fructose. (A) A schematic of fructose metabolism. Distinct metabolic pathways are shown with arrows in different colors: glycolysis and TCA cycle in blue, fructolysis pathway in green, nucleotide and RNA synthesis in yellow, fatty acid and membrane biosynthesis in purple, and amino acids and protein biosynthesis in brown. (B, D) DU145 and HSC-3 cells were stimulated with IL-6 (50 ng/mL) for 24 h, and the level of cellular fructose (B) and GA3P (D) was measured. Data represent the mean and SD from three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001. (C, E-G) DU145 and HSC-3 cells were stimulated with IL-6 (50 ng/mL) for 24 h, and then incubated with 10 µCi [14C]-fructose for 30 min (C) or 1 h (E-G). The level of radiation signal in whole cell lysate (B), total protein (E), total RNA (F) or cell membrane fraction (G) was measured. Data represent the mean and SD from three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001.
Fig 3: IL-6 promotes PD-L1 expression in macrophage both directly and IFN-γ dependently by deregulating protein tyrosine phosphatase, receptor type O (PTPRO). (A) Macrophages isolated from wild-type (WT) and PTPRO KO mice were treated with IFN-γ (50 ng/mL), IL-6 (50 ng/mL), and IFN-γ+IL-6 (50 ng/mL) for 72 hours and the transcription and protein expression of Pd-L1 were determined by using real-time PCR and western blotting, respectively. (B) U937-, THP-1–, U937 PTPRO shRNA–, and THP-1 PTPRO shRNA–derived macrophages were treated with IFN-γ (50 ng/mL), IL-6 (50 ng/mL), or IFN-γ+IL-6 (50 ng/mL each), and the transcription and protein expression of PD-L1 were determined by real-time PCR and western blotting, respectively. (C) Macrophages isolated from WT and PTPRO KO mice were treated with IL-6, and the expression of PD-L1 and PTPRO was detected by real-time PCR and western blotting at different times after IL-6 treatment. (D) U937-, THP-1–, U937 PTPRO shRNA–, and THP-1 PTPRO shRNA–derived macrophages were treated with IL-6, and the expression of PD-L1 and PTPRO was detected by real-time PCR and western blotting at different times after IL-6 treatment. Each experiment was performed in triplicate. Data are presented as the mean±SEM and were analyzed by Student’s t-test (**p<0.01).
Fig 4: The anti-IL-6 antibody reversed the effect of IL-6 on M1 polarization. (A) CCK-8 assay showed the impact of IL-6/anti-IL-6 antibody on the proliferation rate of SW480 and SW620 cells induced by M1 polarization. (B) Transwell assay showed the impact of IL-6/anti-IL-6 antibody on the migration and invasion of SW480 and SW620 cells induced by M1 polarization. Cells were stained with crystal violet. (C) Western blot analysis showing the impact of IL-6/anti-IL-6 antibody on the level of STAT3 phosphorylation induced by M1 polarization. *, anti-IL-6 group vs. M1 polarization group, P<0.05; #, anti-IL-6 group vs. IL-6 group, P<0.05. IL-6, interleukin 6; STAT3, signal transducer and activator of transcription 3; CCK-8, Cell Counting Kit-8.
Fig 5: STAT3 associates with Glut5 promoter and activates Glut5 transcription in response to IL-6. (A-B) DU145 and HSC-3 cells were stimulated with IL-6 (50 ng/mL) for the indicated periods of time. Expression of Glut5 was examined by immunoblot (A) and RT-PCR (B). (C) DU145 and HSC-3 cells were treated with CHX (100 μg/ml) for the indicated periods of time in presence or absence of 50 ng/mL IL-6, and expression of Glut5 was examined by immunoblot. (D) DU145 and HSC-3 cells expressed with STAT3 shRNA were stimulated with IL-6 (50 ng/ml) for 24 h. Immunoblot using indicated antibodies was performed. (E-F) HSC-3 cells were expressed with STAT3 shRNA, SFB-STAT3 WT or SFB-STAT3 Y705F (E). These cells were then stimulated with IL-6 (50 ng/mL) for 24 h (F). Immunoblot using indicated antibodies was performed. STAT3 shRNA targets the non-coding region. (G) HSC-3 cells expressed with STAT3 shRNA and luciferase reporter construct were stimulated with IL-6 (50 ng/mL) for 9 h. Luciferase assay was performed. Data represent the mean and SD from three independent experiments. ***P < 0.001. (H) HSC-3 cells with expression of STAT3 shRNA, SFB-STAT3 WT or SFB-STAT3 Y705F were expressed with luciferase reporter construct, and then stimulated with IL-6 (50 ng/mL) for 9 h. Luciferase assay was performed. Data represent the mean and SD from three independent experiments. **P < 0.01. (I) The putative STAT3-binding site and its franking sequences in Glut5 promoter region is shown. (J) DU145 and HSC-3 cells were stimulated with IL-6 (50 ng/mL) for 2 h. ChIP-PCR was performed. (K) HSC-3 cells expressed with WT and the indicated mutant luciferase reporter constructs were stimulated with IL-6 (50 ng/mL) for 9 h. Luciferase assay was performed. Data represent the mean and SD from three independent experiments. ***P < 0.001; ns, not significant.
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