Journal: Frontiers in Pharmacology

Article Title: Berberine suppresses hepatocellular carcinoma progression by blocking IL-4-JAK1-STAT6-mediated M2 polarization of macrophage

doi: 10.3389/fphar.2025.1734201

Figure Lengend Snippet: Berberine inhibits the activation of the JAK-STAT6 signal pathway induced by H22 conditional medium. (A–F) Relative transcription levels of M2-type signature genes in BMDM, including Arg 1 (A) , Retnla (B) , Il10 (C) , Mrc1 (D) , Tgfb1 (E) , and Chil3 (F) . BMDM were treated with H22 conditional medium for 4 h. (G) Immunoblot analysis of Arg-1 and Retnla protein expression in BMDMs. (H,I) Quantitative analysis of (H) Arg-1 and (I) Retnla protein levels normalized to β-actin. (J) Immunoblot detection of p-STAT6 protein expression in BMDM. (K) Quantitative statistics of p-STAT6/STAT6 are shown. (L) Immunoblot detection of p-JAK1 protein expression in BMDM. (M) Quantitative statistics of p-JAK1/JAK1 are shown. Data (mean ± SEM) are representative of three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001 .

Article Snippet: Primary antibodies were incubated overnight at 4 °C, including: p-STAT6 (56554S, CST), t-STAT6 (9362S, CST), p-JAK1 (3331S, CST), t-JAK1 (3344S, CST), β-actin (8457S, CST), Arg-1 (89872SF, CST), Retnla (Abcam, AB39626).

Techniques: Activation Assay, Western Blot, Expressing

Journal: Frontiers in Pharmacology

Article Title: Berberine suppresses hepatocellular carcinoma progression by blocking IL-4-JAK1-STAT6-mediated M2 polarization of macrophage

doi: 10.3389/fphar.2025.1734201

Figure Lengend Snippet: Berberine inhibits the activation of the JAK-STAT6 signal pathway induced by IL-4. (A–F) Relative transcription levels of M2-type signature genes in BMDMs stimulated by IL-4 (20 ng/mL) for 4 h. Significant changes were observed in Arg1 (A) , Retnla (B), Il10 (C) , Mrc1 (D) , Tgfb1 (E) , and Chil3 (F) . (G,H) Western blotting analysis of p-STAT6 protein expression in BMDMs. Representative immunoblots (G) and quantitative analysis of p-STAT6/STAT6 (H) are shown. (I,J) Content analysis of p-JAK1 protein expression in BMDMs. Representative immunoblots (I) and quantitative analysis of p-JAK1/JAK1 (J) are shown. (K) Co-immunoprecipitation analysis of JAK1-STAT6 interaction in BMDMs under IL-4 (20 ng/mL) stimulation with or without BBR (30 μM) treatment. Data (mean ± SEM) are representative of three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001 .

Article Snippet: Primary antibodies were incubated overnight at 4 °C, including: p-STAT6 (56554S, CST), t-STAT6 (9362S, CST), p-JAK1 (3331S, CST), t-JAK1 (3344S, CST), β-actin (8457S, CST), Arg-1 (89872SF, CST), Retnla (Abcam, AB39626).

Techniques: Activation Assay, Western Blot, Expressing, Immunoprecipitation

Journal: Frontiers in Pharmacology

Article Title: Berberine suppresses hepatocellular carcinoma progression by blocking IL-4-JAK1-STAT6-mediated M2 polarization of macrophage

doi: 10.3389/fphar.2025.1734201

Figure Lengend Snippet: Synergistic therapeutic effects of Berberine and Anti-PDL1 antibody in treating H22 tumor. (A) Schema of the mouse tumor model: Female Bal/bc mice were engrafted with H22 tumor cells (5 × 10 5 ), received intragastric (i.g.) BBR (10 mg/kg), intraperitoneal (i.p.) Anti-PD-L1 antibody (10 mg/kg), or combination treatment. Tumors were removed and analyzed on day 21. (B) Tumor volume was measured every 2 days from day 9 after tumor implantation (n = 6). (C,D) On day 21 after tumor cell implantation, the tumors in mice were removed and weighed. (n = 6). Tumor growth curves (C) and tumor weight (D) are shown. (E–L) Flow cytometry analysis for the percentages of CD45 + lymphocytes in tumor tissues (E) , Percentage of CD3 + cells within the gated CD45 + cells in melanoma tissues (F) , CD3 + CD4 + T cells (G) , CD3 + CD8 + T cells (H) , CD45 + CD11b + Ly6G − Ly6C + M-MDSC (I) , CD45 + CD11b + Ly6G − LY6C − F4/80 + TAMs (J) , CD11b + F4/80 + MHC-Ⅱ + macrophages (K) , and CD11b + F4/80 + CD206 + macrophages (L) , within CD45 + population in tumors. Data (mean ± SEM) are representative of three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001. Schematic diagram illustrating the role of BBR in inhibiting tumor progression. BBR suppresses M2 polarization of tumor-associated macrophages in the tumor microenvironment and enhances the tumor-killing capacity of cytotoxic T lymphocytes, thereby inhibiting tumor progression. Mechanistically, BBR binds to the JAK1 protein in macrophages, inhibiting IL-4-induced phosphorylation of the JAK1-STAT6 signaling axis. This inhibition reduces the secretion of alternative activation genes by macrophages and blocks their pro-tumorigenic functions.

Article Snippet: Primary antibodies were incubated overnight at 4 °C, including: p-STAT6 (56554S, CST), t-STAT6 (9362S, CST), p-JAK1 (3331S, CST), t-JAK1 (3344S, CST), β-actin (8457S, CST), Arg-1 (89872SF, CST), Retnla (Abcam, AB39626).

Techniques: Tumor Implantation, Flow Cytometry, Phospho-proteomics, Inhibition, Activation Assay

Journal: PLoS ONE

Article Title: NK-4 exerts selective regulatory effects on the activation and function of allergy-related Th2 cells

doi: 10.1371/journal.pone.0199666

Figure Lengend Snippet: NHDF were grown in confluent monolayer cultures in 12-well plates and were stimulated with 10 ng/ml IL-4 and 5 ng/ml TNF-α in the presence or absence of varying concentrations of NK-4 for 15 min. Phosphorylation of STAT6 was determined by immunoblotting whole-cell lysates using specific antibodies against the phosphorylated or total STAT6 protein. A representative blot is shown (A). The optical density ratio of phospho-STAT6 to total STAT6 is shown (B). Data from three independent experiments were combined and expressed as the means ± SD. ** p < 0.01 compared with control cultures. Original uncropped and unadjusted Western blots were provided as supplementary files ( and ).

Article Snippet: Membranes were probed with a 1:1000 dilution of anti-phospho-STAT6 (Tyr641) rabbit mAb (C11A12; Cell Signaling Technology, Danvers, MA).

Techniques: Phospho-proteomics, Western Blot, Control

Journal: mBio

Article Title: ROP16-Mediated Activation of STAT6 Suppresses Host Cell Reactive Oxygen Species Production, Facilitating Type III Toxoplasma gondii Growth and Survival

doi: 10.1128/mBio.03305-20

Figure Lengend Snippet: Suppression of STAT6 restricts parasite growth and survival. (A) Replication of WT III in knockdown HFFs at 24 hpi. Mean percentages of parasite vacuoles containing indicated number of parasites. Numbers above stacked bar graph represent the overall mean number of parasites per vacuole. Bars, mean ± SEM. n ≥ 100 vacuoles/well, 3 wells/experiment, 3 experiments. (B) Intracellular survival of WT III in knockdown HFFs 24 hpi expressed as a percentage of initial infection at 6 hpi. HFFs were infected with parasites and stained as in Fig. 2 . Values, mean ± SEM. n = 3 experiments. (C) Quantification of ROS in knockdown HFFs. Each dot = mean ROS value for 1 experiment. n = 100 cells/experiment, 3 experiments. Bars, mean ± SEM. (D) Replication of WT III in WT or STAT6KO mETFs at 24 hpi. Mean percentages of parasite vacuoles containing indicated number of parasites. Numbers above stacked bar graph represent the overall mean number of parasites per vacuole. Bars, mean ± SEM. n ≥ 100 vacuoles/well, 3 wells/experiment, 3 experiments. (E) Intracellular survival of WT III in WT or STAT6KO mETFs at 24 hpi expressed as a percentage of initial infection at 6 hpi. HFFs were infected with parasites and stained as in Fig. 2 . Values, mean ± SEM. n = 3 experiments. (F) Quantification of ROS in WT or STAT6KO mETFs as in Fig. 3 . Each dot = mean ROS value for 1 experiment. n = 100 cells/experiment, 3 experiments. Bars, mean ± SEM. (A and D) Two-way ANOVA, Dunnett’s multiple-comparison test compared to shNT or WT. ****, P ≤ 0.0001. ns = not significant. (B, C, E, and F) One-way ANOVA, Dunnett’s multiple-comparison test compared to shNT or WT. *, P ≤ 0.05; **, P ≤ 0.005; ***, P ≤ 0.0005; ****, P ≤ 0.0001. ns = not significant.

Article Snippet: Western blot analyses were performed using antibodies specific for total STAT3, STAT5a, and STAT6 and for the phosphorylated forms of STAT3 (phospho-Tyr705), STAT5a (phospho-Tyr694), and STAT6 (phospho-Tyr641) (Cell Signaling Technologies; catalog numbers 9139, 94205, 5397, 9145, 9314, and 56554).

Techniques: Knockdown, Infection, Staining, Comparison