Journal: bioRxiv

Article Title: STAT6 mRNA and protein knockdown using multiple siRNA sequences inhibits proliferation and induces apoptosis of the human colon adenocarcinoma cell line, HT-29

doi: 10.1101/462895

Figure Lengend Snippet: (A) STAT6 mRNA level measure. The graphs represent the mean ± SEM of 3 independent experiments. Values were obtained by real-time PCR and results were analysed by ∆∆Ct method for relative quantifications. The fold change is represented on the Y axis, and values are normalized to control cells. (B) STAT6 protein level analysis. The graphs represent the mean of the percentage of STAT6 positive cells ± SEM of 2 independent experiments obtained by flow cytometry. The percentage of STAT6 positive cells is represented on the Y axis. STAT6 siRNAs and non-targeting siRNA were used at 10, 25, 50, 100 and 200 nM as the final concentration. Control cells were non-transfected cells and STAT6 siRNA sequences 1, 2, 3 and 4 and non-targeting siRNA are denoted as STAT6.1, STAT6.2, STAT6.3 and STAT6.4 and NT, respectively.

Article Snippet: Cells were washed twice with PBS/0.5%BSA and stained with anti-STAT6 APC conjugated antibody (Miltenyi Biotec, 130-104-030) (20 μl/10 6 cells) and anti-GAPDH FITC conjugated antibody (Millipore, 130-104-030) (2 μl/ 10 6 cells) for 30 min in the dark at 4°C.

Techniques: Real-time Polymerase Chain Reaction, Control, Flow Cytometry, Concentration Assay, Transfection

Journal: bioRxiv

Article Title: STAT6 mRNA and protein knockdown using multiple siRNA sequences inhibits proliferation and induces apoptosis of the human colon adenocarcinoma cell line, HT-29

doi: 10.1101/462895

Figure Lengend Snippet: (A) Number of live cells measured at day 3, 6 and 8 of culture. The graphs represent the mean ± SEM of 2 independent experiments. (B) The graph shows how cells grew over time and represents the mean ± SEM of the independent experiments shown in A. The number of live cells was calculated as detailed in the material and methods section using NucleoCounter NC-100. STAT6 siRNAs and non-targeting siRNA were used at 100 nM as final concentration. (C) Inverted microscope image taken at day 8 of culture. Control cells were non-transfected cells and STAT6 siRNA sequences 1, 2, 3 and 4 and non-targeting siRNA are denoted as STAT6.1, STAT6.2, STAT6.3 and STAT6.4 and NT, respectively. The percentage of reduction of the number of live cells is calculated by comparison between the mean of NT vs . the mean of STAT6 siRNAs.

Article Snippet: Cells were washed twice with PBS/0.5%BSA and stained with anti-STAT6 APC conjugated antibody (Miltenyi Biotec, 130-104-030) (20 μl/10 6 cells) and anti-GAPDH FITC conjugated antibody (Millipore, 130-104-030) (2 μl/ 10 6 cells) for 30 min in the dark at 4°C.

Techniques: Concentration Assay, Inverted Microscopy, Control, Transfection, Comparison

Journal: bioRxiv

Article Title: STAT6 mRNA and protein knockdown using multiple siRNA sequences inhibits proliferation and induces apoptosis of the human colon adenocarcinoma cell line, HT-29

doi: 10.1101/462895

Figure Lengend Snippet: (A) STAT6 mRNA level measure. The graph represents the mean ± SEM of 6 (Control, NT and STAT6.1) or 3 (STAT6.4) independent experiments obtained by real-time PCR. Results were analysed by ∆∆Ct method for relative quantifications. The fold change is represented by the Y axis, and values are normalized to control cells. (B) STAT6 protein level analysis. The graph represents the mean of the percentage of STAT6 positive cells ± SEM of 6 (Control, NT and STAT6.1) or 5 (STAT6.4) independent experiments obtained by flow cytometry. (C) Representative dot plot and (D) histogram of STAT6 protein analysis by flow cytometry. STAT6 siRNA sequences and non-targeting siRNA were used at 100 nM as the final concentration. Control cells were non-transfected cells and STAT6 siRNA sequences 1 and 4 and non-targeting siRNA are denoted as STAT6.1, STAT6.4 and NT, respectively.

Article Snippet: Cells were washed twice with PBS/0.5%BSA and stained with anti-STAT6 APC conjugated antibody (Miltenyi Biotec, 130-104-030) (20 μl/10 6 cells) and anti-GAPDH FITC conjugated antibody (Millipore, 130-104-030) (2 μl/ 10 6 cells) for 30 min in the dark at 4°C.

Techniques: Control, Real-time Polymerase Chain Reaction, Flow Cytometry, Concentration Assay, Transfection

Journal: bioRxiv

Article Title: STAT6 mRNA and protein knockdown using multiple siRNA sequences inhibits proliferation and induces apoptosis of the human colon adenocarcinoma cell line, HT-29

doi: 10.1101/462895

Figure Lengend Snippet: (A) Number of live cells measured at day 6 of culture. The graph represents the mean ± SEM of 7 (Control, NT and STAT6.1) or 4 (STAT6.4) independent experiments. (B) Number of live cells measured at day 8 of culture. The graph represents the mean ± SEM of 8 (Control, NT and STAT6.1) or 5 (STAT6.4) independent experiments. (C) The graph illustrates how cells grew over time and represents the mean ± SEM of the independent experiments shown in A and B. The number of live cells was calculated as detailed in the material and methods using NucleoCounter NC-100. STAT6 siRNA sequences and non-targeting (NT) siRNA were used at 100 nM as the final concentration. Non-transfected cells served as negative controls and STAT6 siRNA sequences 1 and 4 and non-targeting siRNA are denoted as STAT6.1, STAT6.4 and NT, respectively.

Article Snippet: Cells were washed twice with PBS/0.5%BSA and stained with anti-STAT6 APC conjugated antibody (Miltenyi Biotec, 130-104-030) (20 μl/10 6 cells) and anti-GAPDH FITC conjugated antibody (Millipore, 130-104-030) (2 μl/ 10 6 cells) for 30 min in the dark at 4°C.

Techniques: Control, Concentration Assay, Transfection

Journal: bioRxiv

Article Title: STAT6 mRNA and protein knockdown using multiple siRNA sequences inhibits proliferation and induces apoptosis of the human colon adenocarcinoma cell line, HT-29

doi: 10.1101/462895

Figure Lengend Snippet: (A) Late Apoptosis: percentage of Annexin V and PI positive cells. (B) Total Apoptosis: percentage of Annexin V positive cells. The graphs represent the mean ± SEM of 7 (Control, NT and STAT6.1) or 5 (STAT6.4) independent experiments obtained by flow cytometry. (C) Representative flow cytometry plots. The X axis represents Annexin V and the Y axis, PI fluorescence intensity. Quadrants were set according to cells independently stained with Annexin V or PI. Apoptosis was studied 7 days post-transfection and data were analysed with Flowjo Software. STAT6 siRNA sequences and a non-targeting siRNA sequence were used at 100 nM as the final concentration. Non-transfected cells served as control cells and STAT6 siRNA sequences 1 and 4 and non-targeting siRNA are denoted as STAT6.1, STAT6.4 and NT, respectively.

Article Snippet: Cells were washed twice with PBS/0.5%BSA and stained with anti-STAT6 APC conjugated antibody (Miltenyi Biotec, 130-104-030) (20 μl/10 6 cells) and anti-GAPDH FITC conjugated antibody (Millipore, 130-104-030) (2 μl/ 10 6 cells) for 30 min in the dark at 4°C.

Techniques: Control, Flow Cytometry, Fluorescence, Staining, Transfection, Software, Sequencing, Concentration Assay

Journal: bioRxiv

Article Title: STAT6 mRNA and protein knockdown using multiple siRNA sequences inhibits proliferation and induces apoptosis of the human colon adenocarcinoma cell line, HT-29

doi: 10.1101/462895

Figure Lengend Snippet: (A) STAT6 expression at mRNA level. The graph represents the mean ± SEM of 3 independent experiments. Total mRNA was measured by real-time PCR and results were analysed by the ∆∆Ct method for relative quantifications and values were normalized to control cells. (B) STAT6 expression at protein level. The graph represents the mean ± SEM of 3 independent experiments. Data was analysed using Flowjo Software for MacOS. The percentage of STAT6 positive cells is represented on the Y axis. (C) Representative dot plots and histogram from one set of experiments. STAT6 fluorescence is represented on the X axis. (D) Cell proliferation analysis. Number of live cells measured at day 6 and 8 of culture. The graphs represent the mean ± SEM of 3 independent experiments. The number of live cells was calculated as detailed in the material and methods section using NucleoCounter NC-100.

Article Snippet: Cells were washed twice with PBS/0.5%BSA and stained with anti-STAT6 APC conjugated antibody (Miltenyi Biotec, 130-104-030) (20 μl/10 6 cells) and anti-GAPDH FITC conjugated antibody (Millipore, 130-104-030) (2 μl/ 10 6 cells) for 30 min in the dark at 4°C.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Control, Software, Fluorescence

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

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: Cell reports

Article Title: Mass Cytometry Reveals Global Immune Remodeling with Multi-lineage Hypersensitivity to Type I Interferon in Down Syndrome

doi: 10.1016/j.celrep.2019.10.038

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit monoclonal anti-human phospho-STAT6 (Tyr641) (clone 18) , Fluidigm , Cat#3168012A; RRID:AB_2811103.

Techniques: Recombinant, Mass Cytometry, Software