Fig 1.
STAT6 siRNA sequences 1 and 4 significantly block STAT6 expression.
(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.
Fig 2.
STAT6 siRNA sequences 1 and 4 significantly reduce cell proliferation.
(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.
Fig 3.
STAT6 siRNA sequences 1 and 4 induce apoptosis.
(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.
Fig 4.
STAT6 siRNA sequential transfection is effective in maintaining a reduced number of HT-29 cells.
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.
Fig 5.
JetPEI transfection reagent works for transfecting efficiently STAT6 siRNAs in vitro.
(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.