Journal: PLoS ONE

Article Title: STAT6 knockdown using multiple siRNA sequences inhibits proliferation and induces apoptosis of human colorectal and breast cancer cell lines

doi: 10.1371/journal.pone.0207558

Figure Lengend Snippet: (A) Measure of STAT6 mRNA level in HT-29 cells 24 hours post-transfection. The graph represents the mean ± SEM of multiple independent experiments (n) obtained by real-time PCR. Results were analysed by ΔΔCt 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 at 2, 5 and 7 days post-transfection in HT-29 cells. The graph represents the mean of the percentage of STAT6 positive cells ± SEM of multiple independent experiments (n) obtained by flow cytometry. (C) Measure of STAT6 mRNA level in ZR-75-1 cells 3 days post-transfection. The graph represents the mean ± SEM of multiple independent experiments (n) obtained by real-time PCR. Results were analysed by ΔΔCt method for relative quantifications. The fold change is represented by the Y axis, and values are normalized to control cells. (D) STAT6 protein level analysis after 4 and 7 days of transfection in ZR-75-1 cells. The graph represents the mean of the percentage of STAT6 positive cells ± SEM of multiple independent experiments (n) obtained by flow cytometry. E) Representative histograms (Control: back; NT: grey; STAT6.1: red; STAT6.4: blue) of STAT6 protein analysis at 7 days post-transfection by flow cytometry in HT-29 cells (top) and ZR-75-1 (bottom) cells. 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. The number of independent experiments (n) is set out in the Fig.

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

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

Journal: PLoS ONE

Article Title: STAT6 knockdown using multiple siRNA sequences inhibits proliferation and induces apoptosis of human colorectal and breast cancer cell lines

doi: 10.1371/journal.pone.0207558

Figure Lengend Snippet: (A and B) Number of live HT-29 cells measured at 5 and 7 days post-transfection, respectively. The graphs represent the mean ± SEM of multiple independent experiments (n). (C) The graph illustrates how HT-29 cells grew over time and represents the mean ± SEM of the independent experiments (n) shown in A and B. (D and E) Number of live ZR-75-1 cells measured at 4 and 7 days post-transfection, respectively. The graphs represent the mean ± SEM of multiple independent experiments (n). (F) The graph illustrates how ZR-75-1 cells grew over time and represents the mean ± SEM of the multiple independent experiments (n) shown in D and E. The number of live cells was calculated as detailed in the material and methods using NucleoCounter NC-100. The percentage of reduction of the number of live cells was calculated by comparison between the mean of NT vs . the mean of the transfection with STAT6 siRNA sequences. STAT6 and non-targeting (NT) siRNA sequences were used at 100 nM as the final concentration. Non-transfected cells served as negative control and STAT6 siRNA sequences 1 and 4 and non-targeting siRNA are denoted as STAT6.1, STAT6.4 and NT, respectively. The number of independent experiments (n) is set out in the Fig.

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

Techniques: Transfection, Comparison, Concentration Assay, Negative Control

Journal: PLoS ONE

Article Title: STAT6 knockdown using multiple siRNA sequences inhibits proliferation and induces apoptosis of human colorectal and breast cancer cell lines

doi: 10.1371/journal.pone.0207558

Figure Lengend Snippet: (A) Apoptosis analysis in HT-29 cells. (B) Apoptosis analysis in ZR-75-1 cells. In both cases, the graphs represent from left to right: early (Annexin V + /PI - ), late (Annexin V + /PI + ) and total (Annexin V + ) apoptosis. The graphs represent the mean ± SEM of multiple independent experiments (n) obtained by flow cytometry. (C) Representative flow cytometry plots in HT-29 cells. (D) Representative flow cytometry plots in ZR-75-1 cells. The X axes represent Annexin V and the Y axes represent PI fluorescence intensity. Quadrants were set according to cells independently stained with Annexin V or PI. Apoptosis was studied at 7 days post-transfection in both cell lines. 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. The number of independent experiments (n) is set out in the Fig.

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

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

Journal: PLoS ONE

Article Title: STAT6 knockdown using multiple siRNA sequences inhibits proliferation and induces apoptosis of human colorectal and breast cancer cell lines

doi: 10.1371/journal.pone.0207558

Figure Lengend Snippet: STAT6 siRNA transfection was carried out at day 1 of cell culture with (A) STAT6.1 and (B) STAT6.4 at 100 nM. A second transfection was carried out in both cases with STAT6.1 and STAT6.4 at the same concentration 7 days after the first transfection. The graphs represent the number of live cells over time measured at day 7 and 14 days post-first transfection counted using NucleoCounter NC-100 as detailed in the material and methods section. The mean ± SEM of 3 independent experiments is represented in each graph. 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. The percentage of reduction of the number of live cells was calculated by comparison between the mean of NT vs . the mean of the individual transfection with STAT6 siRNA sequences, and sequential transfection with NT (NT+NT) vs . sequential transfection with STAT6.1 and STAT6.4.

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

Techniques: Transfection, Cell Culture, Concentration Assay, Control, Comparison

Journal: PLoS ONE

Article Title: STAT6 knockdown using multiple siRNA sequences inhibits proliferation and induces apoptosis of human colorectal and breast cancer cell lines

doi: 10.1371/journal.pone.0207558

Figure Lengend Snippet: (A) STAT6 expression at protein level in HT-29 cells at day 2 and 7 post-transfection measured by flow cytometry. The graph represents the mean ± SEM of multiple independent experiments (n). Data was analysed using Flowjo Software. The percentage of STAT6 positive cells is represented on the Y axis. (B) Representative histograms (Control: back; NT: grey; STAT6.1: red; STAT6.4: blue) of STAT6 protein analysis at 7 days post-transfection by flow cytometry in HT-29 cells. (C) Number of live HT-29 cells measured at day 7 post-transfection by NucleoCounter NC100. The graph represents the mean ± SEM of multiple independent experiments (n). (D) The graph illustrates how HT-29 cells grew over time and represents the mean ± SEM of the independent number of experiments shown in C. (E, F and G) The graphs show early (E) (Annexin V + /PI - ), late (F) (Annexin V + /PI + ) and total (G) (Annexin V + ) apoptosis, and represent the mean ± SEM of multiple independent experiments (n) obtained by flow cytometry. Apoptosis was studied at 7 days post-transfection. 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. The number of independent experiments (n) is set out in the Fig.

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

Techniques: Expressing, Transfection, Flow Cytometry, Software, Control, Sequencing, Concentration Assay

Journal: bioRxiv

Article Title: Pro- and anti-inflammatory macrophages adjust UCP2 protein levels based on their intrinsic metabolism and available metabolites

doi: 10.1101/2025.09.08.674165

Figure Lengend Snippet: (A) Scheme of differentiation and polarization of bone marrow-derived macrophages into pro- and anti-inflammatory phenotypes. (B) Representative WB showing STAT1 and STAT6 as well as their phosphorylated forms (pSTAT1 and pSTAT6) in LPS-MΦ and IL4-MΦ, respectively. (C) Representative WB confirming the expression of UCP2 in pro-ad anti-inflammatory macrophages. Recombinant mouse UCP2 (1 ng) and spleen, and skeletal muscle (SkM) were used as positive and negative controls for UCP2 expression, respectively. 20 µg total cell or tissue protein was loaded per lane.

Article Snippet: Antibodies against STAT1 (9172, Cell Signaling Technology, Inc., Massachusetts, USA), p-STAT1 (9167, Cell Signaling Technology, Inc., Massachusetts, USA), STAT6 (9162, Cell Signaling Technology, Inc., Massachusetts, USA), p-STAT6 (9361, Cell Signaling Technology, Inc., Massachusetts, USA), bcl2 (3498, Cell Signaling Technology, Inc., Massachusetts, USA), BAX (2772, Cell Signaling Technology, Inc., Massachusetts, USA), NFKBIA/IkB alpha (H-4) (sc-1643, Santa Cruz Biotechnology Inc., USA), and α-tubulin (801202, Bio Legend Inc., San Diego, USA) were used at dilutions 1:1000, and 1:10000, respectively.

Techniques: Derivative Assay, Expressing, Recombinant

Journal: Molecular immunology

Article Title: Decreasing effects of protein kinase inhibitors on the expression of NOS2 and inflammatory cytokines and on phagocytosis in rat peritoneal macrophages is partly related to repolarization.

doi: 10.1016/j.molimm.2022.11.002

Figure Lengend Snippet: Fig. 6. Phosphorylation of STAT3 and STAT6 proteins in rat macrophages. pSTAT3/STAT3 ratio (a) and protein bands (b., pSTAT3, STAT3 and GAPDH) are shown. JAK/ STAT kinase inhibitors and IL-4 were added to macro phages from casein-treated rats during 24 h culture. IL-4 treatment caused significant (*p < 0.05), while AT and CYT caused strongly significant (***p < 0.001) decrease in phosphorylation of STAT3. Polyclonal anti-pSTAT3 anti body (rabbit, R & D Systems, 1:1000), monoclonal anti- STAT3 antibody (mouse, R & D Systems, 1:2000) and anti-GAPDH (R& D Systems, 1:3000) were used as primary, while HRPO-conjugated goat anti-rabbit (R & D Systems, 1:2000) and goat anti-mouse (R & D Systems, 1:3000) were used as secondary antibodies. c.) Results of cell-based ELISA experiments for pSTAT6 and STAT6 are shown. * *p < 0.01 between res 0′ and cas 0′; §p < 0.05 between casein 0′ and 24 h; ###p < 0.001 for IL-4, AT and CYT treatments compared to casein 24 h as control. Polyclonal anti-pSTAT6 antibody (rabbit, R & D Systems, 1:200), monoclonal anti-STAT6 antibody (mouse, R & D Systems, 1:200) and monoclonal anti-GAPDH (mouse, R & D Sys tems, 1:300) were used as primary, while HRPO- conjugated goat anti-rabbit (R & D Systems, 1:800) and goat anti-mouse (R & D Systems, 1:800) were used as secondary antibodies.

Article Snippet: Fixed cells were then treated with 50 μl rabbit polyclonal anti-phospho-STAT6 antibody (1:200, R & D Systems, MN USA), 50 μl mouse monoclonal anti-STAT6 antibody (1:200, R & D Systems) and 50 μl mouse monoclonal anti-GAPDH antibody (1:300, R & D Systems) for overnight at 4 ◦C.

Techniques: Phospho-proteomics, In-Cell ELISA, Control

Journal: BMC Complementary Medicine and Therapies

Article Title: Anti-cancer effects of genistein supplementation and moderate-intensity exercise in high-fat diet-induced breast cancer via regulation of inflammation and adipose tissue metabolism in vivo and in vitro

doi: 10.1186/s12906-025-04968-x

Figure Lengend Snippet: Effects of GEN, myokines, or their combination on the JAK1/STAT6 pathway in U937 cells. ( A ) U937 cells were treated with GEN, Iri (20 nM), or their combination after PMA and IL-4 treatment. ( B ) U937 cells were treated with GEN, OSM (20 ng/mL), or their combination after PMA and IL-4 treatment. ( a ) Representative pictures were shown. Protein expressions of ( b ) p-JAK1/JAK1 and ( c ) p-STAT6/STAT6 were assessed by Western blot assay. All data are presented as the mean ± standard error of the mean (SEM) from at least three independent experiments. Statistical analysis was performed using one-way analysis of variance (ANOVA), followed by the Newman-Keuls post hoc test. Statistical significance among groups was denoted by different alphabetical letters ( P < 0.05). GEN, genistein; H, breast cancer-induced mice fed a high-fat diet; HG, breast cancer-induced mice fed a high-fat diet with GEN; HE, breast cancer-induced mice fed a high-fat diet with moderate-intensity exercise; HGE, breast cancer-induced mice fed a high-fat diet with GEN and moderate-intensity exercise

Article Snippet: The primary antibodies were used in this study as follows: Bcl-2, Bax, Proliferating cell nuclear antigen (PCNA) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), cleaved caspase-3, total-JAK1, phospho-JAK1, total-STAT6, phospho-STAT6 (Cell Signaling Technology, Danvers, MA, USA), Arg1 (Proteintech, Manchester, UK), CD163, CD68, and β-actin (Abcam, Cambridge, England).

Techniques: Western Blot

Journal: bioRxiv

Article Title: Multidimensional characterization of cellular ecosystems in Hodgkin lymphoma

doi: 10.1101/2025.03.18.643177

Figure Lengend Snippet: (A) Cohort and study design overview. We analyzed whole exome and targeted sequencing data from 114 CHL fresh frozen samples, including 103 patients treated with ABVD-based regimens. Subsequently, we developed tissue microarrays of the cohort and applied whole transcriptome sequencing of CD30+ HRS cells using Nanostring GeoMx technology as well as multiplexed analysis using imaging mass cytometry. Key findings were validated using an independent validation cohort (n=293). (B) Oncoplot showing the most recurrently mutated genes within the CHL samples in the discovery cohort, with clinical annotations. (C) Significantly amplified (red) or deleted (blue) regions across our in-house CHL cohort. (D) Pairwise (Fisher’s exact test) comparison of the incidence of mutations within thymic vs non-thymic CHL cases. The size of the point indicating the strength of association. (E) Frequency of STAT6 mutations in CHL according to age group (YOUNG: < 45 years, OLD: 45 years or older) at diagnosis. YOUNG: < 45 years. Fisher’s exact test was used to determine the significance of differences in observed mutational frequencies. (F) Progression-free survival (PFS) according to mutation and copy number status of STAT6 in young CHL patients (age < 45). P -values were calculated using a log rank test.

Article Snippet: Immunoblotting was performed as previously described( Alig et al ., 2023 ) using the following primary antibodies: STAT1 Rabbit mAb (D1K9Y), STAT3 Mouse mAb (124H6), STAT6 Rabbit mAb (D3H4), AKT(pan) Rabbit mAb (C67E7), Phospho-STAT1 (Y701) Rabbit mAb (58D6), Phospho-STAT3 (Y705) Rabbit mAb (D3A7), Phospho-STAT5 (Y694) Rabbit mAb (C11C5), Phospho-STAT6 (Y641) Rabbit mAb (C11A12), Phospho-AKT (S473) Rabbit mAb (193H12), GAPDH Rabbit mAb (14C10) (all from Cell Signaling Technology, dilution 1:1000), STAT5 Rabbit polyclonalAb (C-17) (dilution 1:5000), CSF2RB (IL-3/IL-5/GM-CSFRβ Mouse mAb (A-3) (dilution 1:100) (Santa Cruz Biotechnology), followed by secondary staining using anti-Mouse (dilution 1:10000; Promega, catalog no. W4021) or Rabbit IgG (H+L) HRP conjugate (dilution 1:5000; Promega, catalog no. W4011), and detected using Amersham ECL Detection Reagents (Cytiva, catalog no. RPN3004).

Techniques: Sequencing, Imaging, Mass Cytometry, Amplification, Comparison, Mutagenesis

Journal: Acta Neuropathologica Communications

Article Title: Multiplex immunoassay characterization and species comparison of inflammation in acute and non-acute ischemic infarcts in human and mouse brain tissue

doi: 10.1186/s40478-016-0371-y

Figure Lengend Snippet: Immune cell composition in infarcts at the stage of liquefactive necrosis in humans and mice. a Representative images of CD3+ T-lymphocyte, CD20+ B-lymphocyte, and CD68+ macrophage/microglia infiltration in human infarcts at the stage of liquefactive necrosis. Scale bars, 10 μm (human, CD3 and CD20 images) and 30 μm (human, CD68 image). b Quantification of CD4+ and CD8+ T-lymphocyte, and CD20+ B-lymphocyte infiltration into the infarcts. c Representative images of CD3+ T-lymphocytes, B220+ B-lymphocytes, and CD68+ macrophages/microglia in the infarcts of C57BL/6 and BALB/c mice at 7 weeks post-stroke. Scale bar,100 μm. d Higher magnification images of CD68+ macrophages/microglia in the infarct to reveal individual cells. Scale bar, 25 μm. e Quantification of CD3+ T-lymphocyte infiltration, B220+ B-lymphocyte infiltration, and CD68 immunostaining in mouse infarcts. **** p < 0.0001 compared to week 1, +++ p < 0.001 compared to BALB/c, ++++ p < 0.0001 compared to BALB/c. f Flow cytometry on cells gated by CD4, using markers of Th1 cells (T-bet and IFNγ), demonstrates that at 7 weeks post-stroke, the CD4+ T cell response in the infarct in C57BL/6 mice is more polarized towards a Th1 response than in BALB/c mice. g Flow cytometry on cells gated by CD4, using markers of Th2 cells (STAT6 and IL-4), demonstrates that at 7 weeks post-stroke, the T cell response in the infarct in BALB/c mice is more polarized towards a Th2 response than in C57BL/6 mice. h There were few CD4+ IL-17+ cells detected in the infarct of both strains. i There were no CD4+ Foxp3 cells detected in the infarct in either strain. j Table showing the % CD4 lymphocytes positive for each marker

Article Snippet: Antibodies included: IFN-γ-CFS, (R&D Systems, Minneapolis, MN), IL-12 Rβ2-APC (R&D Systems), T-bet-PE (eBioscience, San Diego, CA), CD4-Pacific Blue (BioLegend, San Diego, CA), IL-17-Alexa Fluor 700 (BioLegend, San Diego, CA), IL-4 R-CFS (R&D Systems), STAT6-APC, (R&D Systems), IL-5-PE (R&D Systems), CD4-Pacific Blue, and FoxP3-Alexa Fluor 700, (R&D Systems).

Techniques: Immunostaining, Flow Cytometry, Marker