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: Cosmetics

Article Title: Broussonetia papyrifera Promotes Hair Growth Through the Regulation of β-Catenin and STAT6 Target Proteins: A Phototrichogram Analysis of Clinical Samples

doi: 10.3390/cosmetics7020040

Figure Lengend Snippet: Figure 2. Luciferase activity of reporter gene by B. papyrifera. (a) For TCF/LEF reporter gene assay, cells were treated with B. papyrifera (1–40 µg/ml) for 24. The positive control group was treated with minoxidil (0.4 µM) or tofactinib (0.4 µM); (b) IL-4 induced STAT6; and (c) IL-6 induced STAT3 reporter gene assay as determined by luciferase activity. Tofactinib (0.4 µM), a JAKs inhibitor, was treated as a positive control. Each assay is representative for 3 experiments. The asterisk(s) indicate a significant statistical significance (** p < 0.05).

Article Snippet: Polyclonal antibodies against total β-catenin, phospho-specific β-catenin (Thr41/Ser45), STAT6, and phosphor-specific STAT6 (Tyr641) were purchased from Cell Signaling Technology (Beverly, MA, USA) and β-actin antibody, minoxidil, and tofactinb were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Techniques: Luciferase, Activity Assay, Reporter Gene Assay, Positive Control

Journal: Cosmetics

Article Title: Broussonetia papyrifera Promotes Hair Growth Through the Regulation of β-Catenin and STAT6 Target Proteins: A Phototrichogram Analysis of Clinical Samples

doi: 10.3390/cosmetics7020040

Figure Lengend Snippet: Figure 3. Effect of B. papyrifera on the level or activity of proteins implicated in hair growth. (a) The level of p-β-catenin and β-catenin was detected by Western blotting using specific antibodies in hHF DP cells; (b) The level of p-STAT6 and STAT6 was detected by Western blotting using specific antibodies in IL-4-induced HDPCs; (c) The level of p-STAT3 and STAT3 was detected by Western blotting using specific antibodies in IL-6-induced HDPCs. β-Actin protein was used as an internal control. Immunoblot assay was performed as described in the Materials and Methods. Each blot is representative for 3 experiments.

Article Snippet: Polyclonal antibodies against total β-catenin, phospho-specific β-catenin (Thr41/Ser45), STAT6, and phosphor-specific STAT6 (Tyr641) were purchased from Cell Signaling Technology (Beverly, MA, USA) and β-actin antibody, minoxidil, and tofactinb were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Techniques: Activity Assay, Western Blot, Control

Journal: Frontiers in Immunology

Article Title: m6A eraser FTO modulates autophagy by targeting SQSTM1/P62 in the prevention of canagliflozin against renal fibrosis

doi: 10.3389/fimmu.2022.1094556

Figure Lengend Snippet: Cana causes STAT6 degradation through inducing autophagy. (A) HK2 cells were treated with the indicated doses of Cana for 48 h serum-free medium culture and cell viability was determined using MTT assay. (n = 4, * p < 0.05, Cana 0 μM vs. other doses of Cana). (B) HK2 cells were treated with Cana (0–20 μM) for 24 h, and then cell lysates were harvested and subjected to immunoblot analyses with the indicated antibodies. (C) To test its protein stability, the half-life of STAT6 was determined. HK2 cells were left untreated or treated with Cana (20 μM) for 24 h, and CHX (50 μM) was added at different time points. The intensity of STAT6 and GAPDH bands was quantified and plotted against the time after CHX addition. (D) HK2 cells were treated with Cana (0–20 μM) for 24 h and subjected to immunofluorescence staining of LC3. (E) HK2 cells were transfected with a tandem mRFP-GFP-LC3 construct for 24 h and then either left untreated or separately treated with rapamycin (1 μM), BafA1 (100 nM), or Cana (40 μM) for 24 h. Representative micrographs of the indicated cells are shown. (F) The mRNA levels of STAT6, Arg-1, PPARα, and CPT-1α in HK2 cells treated with Cana (0–20 μM) for 24 h were measured by qPCR. (* p < 0.05, Cana 0 μM vs . other doses of Cana). (G–I) HK2 cells were transfected with vector or plasmid of STAT6 overexpression. Followed by 24 h serum-free medium culture, cells were treated with TGF-β1 (5 ng/ml) for another 24 h and cotreated with or without Cana (20 μM). (G) Immunoblot analysis was conducted with the indicated antibodies. (H) Immunoblot analyses were employed to confirm the efficiency of overexpressing STAT6. Representative microphages showed the Oil Red O staining in HK2 cells with the indicated treatments. (I) TG content was determined enzymatically. (n = 4, * p < 0.05, TGF-β1 vs. TGF-β1 with other treatments).

Article Snippet: The following antibodies were used: STAT6 (sc-374021), p-STAT6 (sc-136019), Arg-1 (sc-166920), TGF-β1 (sc-146), α-SMA (sc-53142), FN (sc-18827), LC3I/II (sc-398822), SQSTM1 (sc-28359), CPT-1α (sc-393070), PPARα (sc-398394), and GAPDH (sc-32233) from Santa Cruz Biotechnology.

Techniques: MTT Assay, Western Blot, Immunofluorescence, Staining, Transfection, Construct, Plasmid Preparation, Over Expression

Journal: Frontiers in Immunology

Article Title: m6A eraser FTO modulates autophagy by targeting SQSTM1/P62 in the prevention of canagliflozin against renal fibrosis

doi: 10.3389/fimmu.2022.1094556

Figure Lengend Snippet: Atg7 deficiency vanished the protection of Cana in tubulointerstitial fibrosis. (A) HK2 cells were treated with Cana (0–20 μM) for 24 h. The protein expression of STAT6 and p-STAT6 in nucleus and cytoplasm was separated and determined by immunoblot analyses. (B) HK2 cells were pretreated with MG132 (10 μM) or BafA1 (100 nM) for 4 h and cotreated with or without Cana after being cultured with serum-free medium for 24 h. The cell lysates were subjected to immunoblot analyses with the indicated antibodies. (C–E) WT and Atg7 cKO mice received intragastric administration of saline or Cana (20 mg/kg) after UUO operation. (C) Representative images of H&E, Sirius Red, and IHC staining of kidney sections from the indicated group of mice. Quantification of the relative collagen proportional area from each group of mice was performed. (D) TG content in kidney tissue was measured by a commercial kit. (E) Immunoblot analyses for the protein levels of p-STAT6, STAT6, Arg-1, FN, α-SMA, PPARα, CPT-1α, and GAPDH in kidney from the indicated group with quantification on the right panel (n = 8, * p < 0.05, UUO vs . UUO + Cana).

Article Snippet: The following antibodies were used: STAT6 (sc-374021), p-STAT6 (sc-136019), Arg-1 (sc-166920), TGF-β1 (sc-146), α-SMA (sc-53142), FN (sc-18827), LC3I/II (sc-398822), SQSTM1 (sc-28359), CPT-1α (sc-393070), PPARα (sc-398394), and GAPDH (sc-32233) from Santa Cruz Biotechnology.

Techniques: Expressing, Western Blot, Cell Culture, Saline, Immunohistochemistry

Journal: Frontiers in Immunology

Article Title: m6A eraser FTO modulates autophagy by targeting SQSTM1/P62 in the prevention of canagliflozin against renal fibrosis

doi: 10.3389/fimmu.2022.1094556

Figure Lengend Snippet: FTO was downregulated by Cana to enhance the stability of SQSTM1 mRNA through installing m6A modification of its mRNA. m6A levels of HK2 cells treated with or without Cana (20 μM) were detected by using the EpiQuik™ m6A RNA methylation quantification kit (A) and RNA dot blot analyses (B) . Methylation blue (MB) staining served as a loading control. (C) RNA dot blot analyses of m6A levels in mice from the indicated group. (D) HK2 cells treated with or without Cana (20 μM) were harvested and subjected to qPCR to determined STAT6, Arg-1, SQSTM1, FTO, ALKBH5, METTL14, METTL3, and WTAP mRNA levels. (n = 4, * p < 0.05, Ctrl vs. Cana). (E) HK2 cells treated with Cana (0–20 μM) for 24 h were subjected to detected the protein levels of FTO by immunoblot analyses. (F) Mice received intragastric administration of saline or Cana (20 mg/kg) after UUO treatment. The expression of FTO was determined through IHC staining. (G) HK2 cells were pretreated with MG132 (10 μM) or BafA1 (100 nM) for 4 h and cultured with serum-free medium for 24 h. Then, the cells were left and treated with or without Cana (20 μM) for another 24 h. Immunoblot analyses was performed to detect the protein level of FTO. (H–J, L) HK2 cells were transfected with or without plasmid of FTO overexpression 48 h. Then, cells were treated with or without Cana (20 μM) for another 24 h. (H) The levels of LC3 in HK2 cells from the indicated group were detected through immunofluorescence staining. (I) Immunoblot analysis was performed to determine the protein levels of STAT6, FTO, SQSTM1, LC3I/II, and GAPDH. (J) qPCR analyses of SQSTM1 mRNA stability in HK2 cells from the indicated group (* p < 0.05). (K) RNA immunoprecipitation (RIP)-qPCR analyses revealed the SQSTM1 mRNA level enriched by the FTO antibody. The agarose gel electrophoresis analyses of qPCR products are shown on the panel (* p < 0.05). (L) MeRIP-qPCR assay indicated the m6A modification level of HK2 cells from the indicated group (* p < 0.05). (M) HK2 cells transfected with or without SQSTM1 siRNA for 24 h and then treated with Cana (20 μM) along with TGF-β1 (5 ng/ml) for another 48 h. The efficiency of knockdown SQSTM1 in HK2 cells was confirmed by immunoblot analyses. Representative microphages showed the Oil Red O staining in HK2 cells with indicated treatments.

Article Snippet: The following antibodies were used: STAT6 (sc-374021), p-STAT6 (sc-136019), Arg-1 (sc-166920), TGF-β1 (sc-146), α-SMA (sc-53142), FN (sc-18827), LC3I/II (sc-398822), SQSTM1 (sc-28359), CPT-1α (sc-393070), PPARα (sc-398394), and GAPDH (sc-32233) from Santa Cruz Biotechnology.

Techniques: Modification, Methylation, Dot Blot, Staining, Control, Western Blot, Saline, Expressing, Immunohistochemistry, Cell Culture, Transfection, Plasmid Preparation, Over Expression, Immunofluorescence, RNA Immunoprecipitation, Agarose Gel Electrophoresis, Knockdown

Journal: Frontiers in Immunology

Article Title: m6A eraser FTO modulates autophagy by targeting SQSTM1/P62 in the prevention of canagliflozin against renal fibrosis

doi: 10.3389/fimmu.2022.1094556

Figure Lengend Snippet: Proposed model for the therapeutic action of Cana against renal fibrosis. Cana attenuates renal tubular cells’ FAO disorder and renal fibrosis by SQSTM1/autophagy-mediated STAT6 degradation in an m6A-dependent manner.

Article Snippet: The following antibodies were used: STAT6 (sc-374021), p-STAT6 (sc-136019), Arg-1 (sc-166920), TGF-β1 (sc-146), α-SMA (sc-53142), FN (sc-18827), LC3I/II (sc-398822), SQSTM1 (sc-28359), CPT-1α (sc-393070), PPARα (sc-398394), and GAPDH (sc-32233) from Santa Cruz Biotechnology.

Techniques:

Journal: Immunity

Article Title: Complex Autoinflammatory Syndrome Unveils Fundamental Principles of JAK1 Kinase Transcriptional and Biochemical Function

doi: 10.1016/j.immuni.2020.07.006

Figure Lengend Snippet:

Article Snippet: anti-pSTAT6 149 Sm-conjugated Clone 18 , Fluidigm , Cat No.3149004A.

Techniques: Recombinant, Virus, Molecular Cloning, Blocking Assay, Conjugation Assay, Staining, Western Blot, Lysis, Extraction, Mutagenesis, Isolation, Reverse Transcription, Luminex, Transfection, Amplification, Software, Variant Assay