Fig 1: CAY10526 inhibited the JAK/STAT, TGF-β/Smad3 and PI3K/AKT signaling pathway in Hut78 cells. (A) Western blot showing CAY10526 inhibited the expression of JAK1, JAK2 and pSTAT3 in Hut78 cells. (B) The expression of PI3Kp110, PI3Kp85 and p-AKT protein declined as the increased concentration of CAY10526. (C) CAY10526 inhibited the expression of p-Smad3. (D) The expression of CyclinD1 decreased after exposure to CAY10526 with indicated concentrations. Each protein band intensity was quantified by ImageJ software (mean ± SD, n=3), normalized to loading control GAPDH and compared to the levels of control (0 µM). *, P<0.05 versus control. GAPDH, glyceraldehyde phosphate dehydrogenase; JAK/STAT, Janus kinase/signal transduction and transcription; TGF-β, transforming growth factor-β; PI3K/AKT, phosphatidylinositol 3-kinase/protein kinase B.
Fig 2: ORF3a reduces JAK2 expression via the proteasome pathway. (A) ORF3a reduces the JAK2 level, but not JAK1 and TYK2. HEK293T cells were transfected with empty vector (EV) or ORF3a plasmid, then harvested for immunoblotting at 36 h post-transfection. (B) ORF3a decreases JAK2 in a dose-dependent manner. Cells were transfected with incremental amounts of ORF3a plasmid and then harvested for immunoblotting to determine the JAK2 level. (C) JAK2 mRNA level is not affected by ORF3a expression. HEK293T cells were transfected with EV or ORF3a plasmid. At 24 h after transfection, the cells were harvested for RNA extraction and RT-qPCR. Error bars represent standard errors of the results of repeated experiments. (D) JAK2 reduction in ORF3a-expressed cells is restored by MG132 treatment. HEK293T cells were transfected with EV or ORF3a plasmid. At 30 h after transfection, the cells were treated with MG132 for 6 h and then harvested for immunoblotting with antibodies against JAK2, Flag, and tubulin. Relative levels of JAK2 are shown as folds below the images after normalization with tubulin in densitometry analysis. (E) ORF3a increases JAK2 ubiquitination. The EV or ORF3a transfected cells were lysed with IP lysis buffer, followed by IP with JAK2 antibody. The input and IP products were subjected to immunoblotting with antibodies against ubiquitin (Ub), JAK2, Flag, and tubulin. (F) JAK2 half-life is shortened in the presence of ORF3a. HEK293T cells transfected with ORF3a plasmid were treated with cycloheximide (CHX) and harvested at indicated times (h) for immunoblotting. EV transfected cells were included as a control. Relative levels of JAK2 are shown as folds below the images after normalization with tubulin.
Fig 3: MiR-203 overexpression defends against obesity and improves glucose tolerance. A, B qPCR analysis the mRNA levels of lyn andIFN-? signal pathway in sub-WAT derived from HFD mice compared with normal chow (n = 6/group). CWestern blot analysis of Lyn, Jak1, and Stat1 in sub-WAT derived from HFD mice compared with normal chow. D qPCR analysis of miR-203 expression in Ad-miR-203 injected sub-WAT from mice feeding HFD (n = 8/group) or ND (n = 6/group) compared with Ad-vector injected mice respectively. E Body weightsof Ad-miR-203 injected (twice a week) mice feeding HFD (n = 8/group) or ND (n = 6/group) compared with Ad-Vector injected mice respectively. For Ad-Vector + HFD group vs. Ad-miR-203 + HFD group: *P < 0.05; **P < 0.01. Fsub-WAT weights of Ad-miR-203 injected mice feeding HFD (n = 8/group) or ND (n = 6/group) compared with Ad-Vector injected mice respectively. G, H GTT and ITT assays were conducted among Ad-miR-203 and Ad-Vector injected mice feeding HFD (n = 8/group) or ND (n = 6/group). For Ad-Vector + HFD group vs. Ad-miR-203 + HFD group: *P < 0.05; **P < 0.01; ***P < 0.001. I Western blot analysis of p-Akt and Akt in Ad-miR-203 injected sub-WAT (twice a week) from mice feeding HFD or ND compared with Ad-Vector injected mice respectively. J Western blot analysis of Lyn and Stat1 in Ad-miR-203 injected sub-WAT from mice feeding HFD or ND compared with Ad-vector injected mice respectively. K Serum levels of IFN-? among Ad-miR-203 and Ad-Vector injected mice feeding HFD were examined by ELISA kit (n = 8/group). L A working model for the cAMP-miR-203-IFN-? network.*P < 0.05, **P < 0.01, and ***P < 0.001 by Student's t-test. Data presented as mean ± s.e.m.
Fig 4: Molecular models of Palbinone (PB) binding to its predicted protein targets and the anti-inflammatory effect of PB by inhibiting JAK1 in HISF cells. (A) JAK1 binding PB; (B) JAK2 binding PB; (C) PB binding to JAK1 in ATP binding sites LYS908; (D) PB binding to JAK1 in ATP binding sites LYS908. HISF cells were transfected with NC, OE-JAK1 by lentiviral vector. After HISF cells were treated with TNF-α (20 ng/ml) for 24 h, they were incubated with PB (10 μg/ml) for 6 h. The expression of JAK1 (E,F), p-JAK1 (E,G), STAT1 (E,H) and p-STAT1 (E,I) from the indicated group were detected by Western blot assay. The mRNA levels of JAK1 (J) and IL-6 (K) from the indicated group were detected by qPCR. Results were mean ± SD for three individual experiments. *p < 0.05, **p < 0.01.
Fig 5: Fenofibrate re-established macrophages to enhance their antitumor function by activating the PPAR-α-STAT1 and FAO positive feedback loop pathways. (A) Immunofluorescence staining for the expression of cytokines IL-1β and iNOS in Co-CM1, Con-CM with and without FF treatment, as well as Co-CM1 (siPPAR-α) + FF groups. (B) SIRT1, JAK1-STAT1, and FAO pathways were assessed by western blot analysis in CTRL and Co-CM1 groups. (C) ELISA of the IL-12 and IL-23 secretion by macrophages in Co-CM1 and, Con-CM with and without FF treatment. *p < 0.05, **p < 0.01. (D) Elucidation of tumor cells proliferation from culture medium of different conditioned macrophages. (E) Proliferation of MDA-MB-231 cells treated with conditioned media from different macrophages. ***p < 0.001. (F) Clone formation of MDA-MB-231 cells cultured in supernatant from different macrophages.
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