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: Blood

Article Title: Characterization of DLBCL with a PMBL gene expression signature.

doi: 10.1182/blood.2020007683

Figure Lengend Snippet: Figure 5. IL4R mutations in nm-PMBLsig1 tumors induce JAK-STAT activation. (A) Mutational pattern of IL4R (UniProtKB-P24394) in nm-PMBLsig1 tumors (top) and other DLBCL tumors (bottom) from the BCC cohort specified according to receptor domains (extracellular, transmembrane, or cytoplasmic). Colors and shapes of lollipops represent the DLBCL90 NanoString assay tiered classification of samples (see Figure 1) and type of mutation (circle, missense or in-frame indel; diamond, truncating), respectively. The plot was created using R package TrackViewer (version 1.24.0). (B-G) Ectopic expression of WT IL4R and mutants in HEK Blue IL-4/IL-13 (Invivogen) (B-C) and DEV cells (D-G). (B) Quantification of pSTAT6-dependent expression of secreted embryonic alkaline phosphatase (SEAP) in supernatant. (C-D) Immunoblot for pSTAT6, STAT6, IL4R, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). (E-F) CCL17 and CD23 messenger RNA expression measured by quantitative polymerase chain reaction. (G) IL4R surface expression measured by flow cytometry. Data are means 6 standard deviation of 5, 4, 7, and 7 experiments in panels A, E, F, and G, respectively; significance was evaluated using a 1-sample Student t test. *P , .05, **P , .01, ****P , .0001.

Article Snippet: Western blotting and flow cytometry were performed as previously described.31 Briefly, membranes were probed with the following primary antibodies at 1:1000 dilution ratio unless stated otherwise: phosphorylated STAT6 (pSTAT6; 9364; Cell Signaling Technology), STAT6 (ab32108; Abcam), and IL4 receptor (IL4R; sc-28361; 1:500; Santa Cruz Biotechnology).

Techniques: Activation Assay, Mutagenesis, Expressing, Western Blot, RNA Expression, Real-time Polymerase Chain Reaction, Cytometry, Standard Deviation

Journal: Frontiers in Immunology

Article Title: Major vault protein regulates tumor-associated macrophage polarization through interaction with signal transducer and activator of transcription 6

doi: 10.3389/fimmu.2023.1289795

Figure Lengend Snippet: MVP interacts with STAT6 and JAK1. (A) HA-tagged MVP and Flag-tagged STAT6 were co-transfected into HEK293T at 36 h post-transfection, and the cells were collected for Co-immunoprecipitation (Co-IP) and immunoblot analyses. (B) BMDMs were cultured in SF medium overnight after the formation of adherent cells. The next day, cells were stimulated with medium or IL-4 for 30 min and collected for Co-IP and immunoblot analyses. (C) HEK293T cells were transfected with pEGFP-MVP and DsRed-STAT6 for 36 h and then collected for IF assays. Representative image of MVP (green) and STAT6 (red), Scale bar, 10 µm (left panel). The quantitative analysis of co-localization using Image J (right panel). (D) Schematic diagram of structural domains and truncated constructs of STAT6 full-length (FL) (upper panel). HEK293T were co-transfected with HA-tagged MVP and truncated mutants of Flag-tagged STAT6 for 36 h. Then cells were collected for Co-IP and immunoblot analyses (lower panel). (E) Experiments were performed similarly to those in (D) , except the indicated truncated constructs of MVP were used. (F) Flag-JAK1 and HA-MVP were co-transfected into HEK293T for 36 h, and then the cells were harvested for Co-IP and immunoblot analyses. (G) BMDMs were stimulated with or without IL-4 for 30 min and collected for Co-IP and immunoblot analyses. (H) HEK293T cells were transfected with indicated plasmids for 36 h prior to Co-IP and immunoblot analyses. (I) Wt and Mvp -/- BMDMs were stimulated with medium or IL-4 for 30 min prior to Co-IP and immunoblot analyses. All protein abundance analyses were performed using Image J. All experiments were repeated at least three times with similar results. See also Supplementary Figure 7 .

Article Snippet: Antibodies for phospho-STAT6 (Tyr641) (CSB-PA000625) and phospho-STAT1 (Tyr641) (CSB-PA050162) were from Cusabio.

Techniques: Transfection, Immunoprecipitation, Co-Immunoprecipitation Assay, Western Blot, Cell Culture, Construct, Quantitative Proteomics

Journal: Frontiers in Immunology

Article Title: Major vault protein regulates tumor-associated macrophage polarization through interaction with signal transducer and activator of transcription 6

doi: 10.3389/fimmu.2023.1289795

Figure Lengend Snippet: MVP enhances the phosphorylation and nuclear translocation of STAT6. (A) Wt and Mvp -/- BMDMs were stimulated with or without IL-4 at the indicated times before western blot analysis. (B) Raw264.7 cells were transfected with Flag-tagged MVP or vector for 36 h, then stimulated with phosphate-buffered saline (PBS) or IL-4 for 30 min. Immunoblot analyses were performed with the indicated antibodies. (C) Wt and Mvp -/- BMDMs were stimulated with IL-4 for the indicated time. The whole cell lysates (WCL), cytosolic and nuclear extracts were prepared and subjected to western blot analyses. Lamin B and GAPDH were used as nuclear and cytosolic fractions markers, respectively. (D) Experiments were performed similar to those in (C) , except Raw264.7 cells were transfected with Flag-MVP for 36 h. (E) IF of STAT6 in Wt and Mvp -/- PMs stimulated with IL-4. Representative image of STAT6 (green). Scale bar, 20 µm (left panel). The percentage of nuclear STAT6 positive cell numbers was counted (right panel). All protein abundance analyses were performed using Image J. All experiments were repeated at least three times with similar results. Data are expressed as means ± SEM, n = 3, two-tailed Student’s t-test. (**P < 0.01). See also Supplementary Figure 8 .

Article Snippet: Antibodies for phospho-STAT6 (Tyr641) (CSB-PA000625) and phospho-STAT1 (Tyr641) (CSB-PA050162) were from Cusabio.

Techniques: Phospho-proteomics, Translocation Assay, Western Blot, Transfection, Plasmid Preparation, Saline, Quantitative Proteomics, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: Major vault protein regulates tumor-associated macrophage polarization through interaction with signal transducer and activator of transcription 6

doi: 10.3389/fimmu.2023.1289795

Figure Lengend Snippet: MVP-promoted M2-TAMs polarization and tumorigenesis in HCC. In the tumor microenvironment of hepatocellular carcinoma, JAK1 recruits MVP and STAT6, leading to ternary complex formation. Then, STAT6 is phosphorylated and translocated from the cytosol to the nucleus. As a result, STAT6 binds to the promoter of M2 genes, leading to M2 polarization and M2-TAMs infiltration.

Article Snippet: Antibodies for phospho-STAT6 (Tyr641) (CSB-PA000625) and phospho-STAT1 (Tyr641) (CSB-PA050162) were from Cusabio.

Techniques:

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