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: Inflammatory Bowel Diseases

Article Title: IL-6 Mediates the Intestinal Microvascular Thrombosis Associated with Experimental Colitis

doi: 10.1097/mib.0000000000000656

Figure Lengend Snippet: FIGURE 6. Effects of blockade of the alpha (IL-6Ra) and beta (gp130) subunits of the IL-6 receptor on DSS colitis-enhanced thrombus formation in colonic arterioles. *P , 0.05 compared with corresponding WT-H2O group, #compared with WT-DSS group, and +compared with WT-DSS vehicle group.

Article Snippet: Rat antimouse IL-6Ra (CD126)-blocking antibody was purchased from Angio-Proteomie (Boston, MA).

Techniques:

Journal: Cellular and Molecular Immunology

Article Title: The kinase p38α functions in dendritic cells to regulate Th2-cell differentiation and allergic inflammation

doi: 10.1038/s41423-022-00873-2

Figure Lengend Snippet: p38α deletion in DCs promotes Th2 priming and allergic inflammation during the sensitization phase. a – c WT and p38α ΔDC mice were sensitized with HDM on Days 0–2 and challenged with HDM on Days 14–16. Mice were sacrificed for analysis on Day 17. ELISA analysis of IL-4, IL-5, IL-13, IL-17 and IFNγ production in the BALF ( a ) ( n = 3–4). The percentages of IL-4 + , IL-5 + , IL-13 + , IL-17 + and IFNγ + cells in CD4 + T cells were determined by intracellular staining ( b , c ) ( n = 13). Percentages of GATA3 + cells in CD4 + T cells ( d ) ( n = 5). e – h WT and p38α ΔDC mice were sensitized with HDM for 3 days and analyzed 7 days later. Total cell number in the BALF ( e ) ( n ≥ 14). The percentage and cell number of eosinophils were detected by flow cytometry ( f ) ( n ≥ 14). The percentages of IL-4 + , IL-5 + , IL-13 + , IL-17 + and IFNγ + cells in CD4 + T cells were determined by intracellular staining ( g ) ( n ≥ 19). ELISA analysis of ex vivo-isolated mLN cells restimulated with or without HDM for 72 h ( h ) ( n = 3–4). * P < 0.05; ** P < 0.01; ns, not significant. Data are representative of three ( a , d , h ) independent experiments or pooled from three ( b – c , e and f ) or four ( g ) experiments with consistent results. Student’s t test ( c – g ) or two-way ANOVA ( a and h ) was performed, and the data are presented as the mean ± SEM

Article Snippet: For intracellular staining (ICS), cells were stimulated with PMA (Sigma–Aldrich) and ionomycin (Sigma–Aldrich) in the presence of GolgiStop (BD Biosciences) for 5 h and then stained according to the manufacturer’s instructions (BD Biosciences) with antibodies against ms IL-4 (11B11; eBioscience), ms IL-5 (TRFK5; BD Biosciences),ms IL-13 (eBio13A; eBioscience), ms IFNγ (XMG1.2; eBioscience), and ms IL-17 (eBio17B7; eBioscience).

Techniques: Enzyme-linked Immunosorbent Assay, Staining, Flow Cytometry, Ex Vivo, Isolation

Journal: Cellular and Molecular Immunology

Article Title: The kinase p38α functions in dendritic cells to regulate Th2-cell differentiation and allergic inflammation

doi: 10.1038/s41423-022-00873-2

Figure Lengend Snippet: p38α signaling in cDC1s regulates Th2 responses upon HDM treatment. a Flow cytometry-sorted lung DCs from HDM-treated WT and p38α ΔDC mice were cocultured with naïve OT-II CD4 + T cells in the presence of OVA 323-339 for 72 h. ELISA analysis of IL-4, IL-5, IL-13, IL-17 and IFNγ production ( n = 3). b Analysis of Il4 , Il5 , Il13 , Gata3 , Il10 and Il9 mRNA expression in OT-II CD4 + T cells ( n = 4). c , d WT and p38α ΔcDC mice were sensitized and challenged with HDM to induce asthma ( n = 3). Percentages and cell numbers of eosinophils in the BALF and lung tissues ( c ). Percentages of IL-4, IL-5, IL-13 and IL-17 in lung CD4 + T cells ( d ). e mRNA expression of Il4 , Il13 , Gata3 and Tbx21 in OT-II CD4 + T cells activated by WT or p38α-deficient lung cDC1 or cDC2 subsets from HDM-treated mice in the presence of OVA 323-339 ( n = 2–3, 1 DC subset sample pooled from at least 3 mice). f and g WT and p38α ΔcDC1 mice were sensitized and challenged with HDM to induce asthma ( n = 3–7). Airway resistance was measured ( f ). The cell numbers of IL-4 + CD4 + T cells and IL-13 + CD4 + T cells in lung tissues were measured ( g ). h and i WT, p38α ΔDC , IRF8 ΔDC and IRF8/p38α ΔDC mice were sensitized and challenged with HDM to induce asthma ( n = 3–6). The numbers of eosinophils in the BALF and lung tissues ( h ) and the numbers of IL-4 + CD4 + T cells and IL-13 + CD4 + T cells in lung tissues were measured ( i ). * P < 0.05; ** P < 0.01; ns, not significant. Data are pooled from three ( a and b ) or two ( h and i ) experiments with consistent results or representative of three ( c – e ) or two ( f and g ) experiments with consistent results. Student’s t test ( a – d , g – i ) or two-way ANOVA ( e and f ) was performed, and the data are presented as the mean ± SEM

Article Snippet: For intracellular staining (ICS), cells were stimulated with PMA (Sigma–Aldrich) and ionomycin (Sigma–Aldrich) in the presence of GolgiStop (BD Biosciences) for 5 h and then stained according to the manufacturer’s instructions (BD Biosciences) with antibodies against ms IL-4 (11B11; eBioscience), ms IL-5 (TRFK5; BD Biosciences),ms IL-13 (eBio13A; eBioscience), ms IFNγ (XMG1.2; eBioscience), and ms IL-17 (eBio17B7; eBioscience).

Techniques: Flow Cytometry, Enzyme-linked Immunosorbent Assay, Expressing

Journal: Cellular and Molecular Immunology

Article Title: The kinase p38α functions in dendritic cells to regulate Th2-cell differentiation and allergic inflammation

doi: 10.1038/s41423-022-00873-2

Figure Lengend Snippet: p38α signaling in lung cDC1s regulates Th2-cell differentiation by modulating IL-12 expression. a GSEA of cDC1s. b IL-12p40 and ( c ) IL-12p35 expression in HDM-treated WT and p38α-deficient lung DC subsets analyzed by flow cytometry ( n = 4). d mRNA expression of Il4 and Il13 in CD4 + T cells activated with WT or p38α-deficient lung cDC1 or cDC2 subsets from HDM-treated mice with or without IL-12 for 3 days ( n = 2–3, 1 DC subset sample pooled from at least three mice). e – h WT and p38α ΔDC mice were sensitized with HDM on Days 0–2 and challenged with HDM on Days 14–16, and then IL-12p70 was i.n . administered during HDM treatment. Mice were sacrificed for analysis on Day 17 ( n = 3–4). Airway resistance was measured ( e ). Eosinophil infiltration in the BALF and lung tissues was detected by flow cytometry and quantified ( f and g ). The concentrations of IL-4, IL-5 and IL-13 in the BALF were detected by ELISA ( h ). ** P < 0.01; ns, not significant. Data are representative of three ( b – d and f – h ) or two ( e ) independent experiments. Student’s t test ( b , c , g and h ) or two-way ANOVA ( d and e ) was performed, and the data are presented as the mean ± SEM

Article Snippet: For intracellular staining (ICS), cells were stimulated with PMA (Sigma–Aldrich) and ionomycin (Sigma–Aldrich) in the presence of GolgiStop (BD Biosciences) for 5 h and then stained according to the manufacturer’s instructions (BD Biosciences) with antibodies against ms IL-4 (11B11; eBioscience), ms IL-5 (TRFK5; BD Biosciences),ms IL-13 (eBio13A; eBioscience), ms IFNγ (XMG1.2; eBioscience), and ms IL-17 (eBio17B7; eBioscience).

Techniques: Cell Differentiation, Expressing, Flow Cytometry, Enzyme-linked Immunosorbent Assay