Fig 1: Schematic of the action of tumor-derived exosomal circSAFB2 in mediating M2 macrophage polarization through the miR-620/JAK1/STAT3 axis to promote renal cell carcinoma metastasis.
Fig 2: As3+ activates JAK1 and JAK2: BEAS-2B cells were starved overnight then treated with the indicated concentrations of As3+ for 6 h. BEAS-2B cell lysates were prepared following As3+ treatment and immunoblotted on PVDF membranes. Membranes were probed with the indicated antibodies. The levels of protein expression were confirmed by Western blotting.
Fig 3: RCC-derived exosomal circSAFB2 induces M2 macrophage polarization through the miR-620/JAK1/STAT3 axis. (A, B) Schematic representation of the 3'- ‘UTR’ of JAK1 with the predicted target site for miR-620. The mutant site of JAK1 3'-UTR is indicated (without line); (C) Luciferase reporter analysis was performed to examine the binging capacity between miR-620 and JAK1. Reporter constructs containing JAK1wt or JAK1mut at the predicted miR-620 target sequences were co-transfected into macrophages, along with miR-620 mimics or miR-NC; (D, E) Western blotting and densitometric analysis of JAK1 expression in macrophages after transfection with miR-NC or miR-620 mimics; (F, G) Western blotting and densitometric analysis of JAK1 and STAT3 expression in macrophages after transfection with JAK1 siRNA; (H–K). (H, I) Western blotting and densitometric analysis value of JAK1 and STAT3 expression in macrophages after treatment with 769-P-derived exosomes (Exo-769-P), along with miR-NC or miR-620 mimics. (J, K) Western blotting and densitometric analysis value of JAK1 and STAT3 expression in macrophages after treatment with ACHN-derived exosomes (Exo-ACHN), along with miR-NC or miR-620 mimics; (L). qRT-PCR analysis of M2 macrophages (IL-1RA, CD163, CD206 and CCL18) after indicated treatment (Exo-769-P, Exo-769-P + si-circSAFB2, Exo-769-P + si-circSAFB2 pool + miR-620 int, Exo-769-P + si-circSAFB2 pool + miR-620 int + si-JAK1); (M). qRT-PCR analysis of M1 macrophages (TNF-a, IL-1ß and MCP-1) after indicated treatment; (N). ELISA analysis of cytokine released from M2 macrophages (IL-1RA and CCL18) and M1 macrophages (TNF-a and IL-1ß). All data indicate mean ± SD; n=3, **P<0.01, ***P<0.001. ns, no significance.
Fig 4: Type I interferon (IFN) increased expression of OAS2, OAS3, OASL, and inflammatory factors in T cells. CD4+ T cells were treated with IFN-a-2a. A, IFN-a-2a had no significant effect on the cell viability within 24 h. B, Western blot was performed to evaluate phosphorylation levels of Jak1 and Stat3. C, PCR was performed to evaluate OAS2, OAS3, and OASL expression in T cells. D, PCR was performed to evaluate expression of inflammatory factors in T cells. Data are reported as means±SD. *P<0.05, **P<0.01 vs control (Student’s t-test).
Fig 5: A. fumigatus–stimulated DCs activates JAK/STAT through TSLP. (A) DCs were treated with A. fumigatus and then co-cultured with CD4+ T cells for 4 days, a Western blot analysis was performed to determine the protein levels of RORγt, p-JAK1, JAK1, p-JAK2, JAK2, p-STAT3, STAT3, and β-actin in the CD4+ T cells. (B) CD4+ T cells were cultured with rmTSLP (100 ng/mL) for 3 days, protein levels of RORγt, p-JAK1, JAK1, p-JAK2, JAK2, p-STAT3, STAT3, and β-actin were analyzed by western blot. Quantification of relative protein levels in (A) and (B) were shown in (C) and (D), respectively. Cells were divided into four groups and were treated as described in Figure 4C. (E) Western blot analysis of the protein levels of RORγt, p-JAK1, JAK1, p-JAK2, JAK2, p-STAT3, STAT3, and β-actin in CD4+ T cells. (F) CD4+ T cells were incubated with TLSPR siRNA (80 nM) or NC siRNA for 24 hours, then co-cultured with A. fumigatus–stimulated DCs for 4 days. A Western blot was performed to detect the protein levels of RORγt, p-JAK1, JAK1, p-JAK2, JAK2, p-STAT3, STAT3, and β-actin in CD4+ T cells. Quantification of relative protein levels in (E) and (F) were shown in Supplementary Figure S4C and D. (Data are mean ± SEM, *P < 0.05, **P < 0.01, n = 3).
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