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