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: Journal of Neuroinflammation

Article Title: Murine astrocytes produce IL-24 and are susceptible to the immunosuppressive effects of this cytokine

doi: 10.1186/s12974-019-1444-1

Figure Lengend Snippet: IL-24 augments the expression of suppressive cytokine signaling components by murine astrocytes and limits inflammatory cytokine release by these cells. a Astrocytes were untreated or treated with recombinant IL-24 (10, 30, or 100 ng/mL) for 30 min and the presence of phosphorylated STAT3 and STAT1 was determined by immunoblot analysis. Expression of β-actin is shown as a loading control and these immunoblots are representative of two separate experiments. b Astrocytes were untreated or treated with recombinant IL-24 (10, 30, or 100 ng/mL) for 2 or 4 h, and SOCS3 mRNA expression was determined by semi-quantitative RT-PCR. Expression of the housekeeping gene product GAPDH is shown, and relative SOCS3 expression was determined by densitometric analysis and normalized to untreated cells. Data is expressed as the mean ± the SEM of three independent experiments, and an asterisk indicates a statistically significant difference from unchallenged cells at each time point ( p < 0.05). c Astrocytes were untreated or treated with recombinant IL-24 (10, 30, or 100 ng/mL) for 8 h prior to immunoblot analysis for SOCS3 protein expression. Expression of the housekeeping gene β-actin is shown and relative SOCS3 protein expression was determined by densitometric analysis normalized to untreated cells. Asterisks indicate statistically significant differences from unchallenged cells (p < 0.05). d Astrocytes were untreated or treated with IL-24 (0.5, 3, 10, 30, or 100 ng/mL) for 4 h prior to challenge with bacterial LPS (5 ng/mL) or vehicle control for 12 h, and IL-6 secretion was determined by specific capture ELISA. Asterisks indicate a statistically significant difference ( p < 0.05) from similarly challenged cells in the absence of IL-24 ( n = 3). e Primary astrocytes were untreated or treated with recombinant IL-24 (10, 30, or 100 ng/mL) for 4 h prior to being uninfected or infected with Nm for 48 h before cell viability analysis via MTS assay. Data is presented as the mean absorbance ± SEM for three experiments. 0.1% Triton X-100 was used as a positive control, and an asterisk indicates a statistically significant difference from unchallenged cells in the absence of IL-24 ( p < 0.05)

Article Snippet: Commercially available Duoset® ELISA kits were used to measure IL-10 and TNF-α secretion (R&D Systems), while murine IL-6 secretion was measured using a rat anti-mouse IL-6 capture antibody (Clone MP5-20F3) and a biotinylated rat anti-mouse IL-6 detection antibody (Clone MP5-C2311) (BD Biosciences).

Techniques: Expressing, Recombinant, Western Blot, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Infection, MTS Assay, Positive Control