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:

Article Title: Insulin receptor substrate-1/2 mediates IL-4-induced migration of human airway epithelial cells

doi: 10.1152/ajplung.90453.2008

Figure Lengend Snippet: A: phosphorylation of STAT6 in AEC after injury and treatment with IL-4. Cells grown in ALI culture for 3 wk were injured and treated immediately with 10 ng/ml IL-4 or control vehicle only. Cell lysates collected over 2 h were resolved by Western blot for total and phospho-STAT6. Image is representative of 3 experiments. B: phosphorylation of STAT6 in AEC grown in ALI culture for 3 wk after injury and treatment with 10 ng/ml IL-4, 10 ng/ml IL-13, 10 ng/ml IL-1β, or 20 ng/ml TNFα. Cell lysates 30 min after injury and treatment were resolved by Western blot for total and phospho-STAT6. Image is representative of 3 experiments.

Article Snippet: A truncated STAT6 cDNA was generated starting with pCMV6-XL-5-STAT6 (acc. no. NM 003153, OriGene).

Techniques: Western Blot

Journal:

Article Title: Insulin receptor substrate-1/2 mediates IL-4-induced migration of human airway epithelial cells

doi: 10.1152/ajplung.90453.2008

Figure Lengend Snippet: A: migration of differentiated AEC in response to IL-4: effect of IFNγ. Cells were grown under ALI culture conditions for 3 wk and injured using a rubber stylette, and migration was followed for 24 h by digital photomicroscopy. N = 5–8 experiments in each group collected from 2 normal donors. Statistical significance of difference was analyzed by 1-way ANOVA (*P = 0.05; †P = 0.02 vs. control). Data are expressed as means ± SE. B: STAT6 phosphorylation after treatment with 10 ng/ml IL-4 alone or combined with 1–100 ng/ml IFNγ. Cells in ALI culture × 3 wk were treated as noted for 30 min. Image shown is representative of 2 experiments.

Article Snippet: A truncated STAT6 cDNA was generated starting with pCMV6-XL-5-STAT6 (acc. no. NM 003153, OriGene).

Techniques: Migration

Journal:

Article Title: Insulin receptor substrate-1/2 mediates IL-4-induced migration of human airway epithelial cells

doi: 10.1152/ajplung.90453.2008

Figure Lengend Snippet: Blocking IL-4-accelerated migration of differentiated AEC by expression of a truncated, dominant negative STAT6 cDNA. A: expression of GFP as a marker for successful infection with lentivirus (LV) containing cDNA. Undifferentiated AEC were infected with either LV pLL3.7-STAT6-tr + LV pLL3.7-GFP control in 5:1 ratio (STAT6-tr), LV pLL3.7-GFP control alone (EV control), or were sham-infected. GFP fluorescence was examined 3 wk after conversion to ALI culture. Bar, 20 μm. B: abundance of STAT6 and STAT6-tr protein after infection as in A. Protein lysates collected from cells in ALI culture × 3 wk were resolved for total STAT6 by Western blot. Lane loading was confirmed by rehybridization with an antibody directed against actin. The antibody used recognizes both STAT6 and STAT6-tr. C: abundance of STAT6 and STAT6-tr protein in cell nuclei after infection as in A. Cells in ALI culture × 3 wk after infection were treated with 10 ng/ml IL-4 or control diluent for 6 h, after which nuclear proteins were collected and resolved for total STAT6 by Western blot. Increased abundance of STAT6 is seen in nuclear protein extracts after IL-4 treatment in both sham-infected and EV control cells but not in cells infected with pLL3.7-STAT6-tr. D: migration of AEC after infection with LV pLL3.7-STAT6-tr-cDNA. Cells were grown in ALI culture × 3 wk after infection and then injured using a rubber stylette. Cells were then treated with 10 ng/ml IL-4 or sham diluent, and migration was followed for 24 h by digital photomicroscopy. N = 8 experiments in each group. Statistical significance of difference was analyzed by 2-tailed t-test (*P = 0.01 vs. control at 24 h). Data are expressed as means ± SE. E: migration of AEC after infection with LV pLL3.7-GFP control (EV control). Cells were grown in ALI culture × 3 wk after infection and then injured using a rubber stylette. Cells were then treated with 10 ng/ml IL-4 or sham diluent, and migration was followed for 24 h by digital photomicroscopy. N = 7–8 experiments in each group. Statistical significance of difference was analyzed by 2-tailed t-test (*P = 0.01 vs. control at 24 h). Data are expressed as means ± SE.

Article Snippet: A truncated STAT6 cDNA was generated starting with pCMV6-XL-5-STAT6 (acc. no. NM 003153, OriGene).

Techniques: Blocking Assay, Migration, Expressing, Dominant Negative Mutation, Marker, Infection, Fluorescence, Western Blot