Effect of SRSF1 knockdown on expression, stability and translation of Mcl-1_L in MCF-7 and JAR cells.

<p>(<b>A</b>) MCF-7 and (<b>B</b>) JAR cells were transfected with SRSF1 (1 and 2) and negative control (NC) siRNA. Three days after transfection cells were treated with 35 ug/ml cycloheximide and protein samples were collected after 0, 0.5, 1, 1.5, 2, 3, 5 and 7 hours to assess protein stability. Mcl-1_L protein levels were then measured by immunoblotting. (<b>C</b>) MCF-7 cells were transfected with SRSF1 (1 and 2), GAPDH (G) and negative control (NC) siRNA, or treated with vehicle (lipid) only (V), or were untreated (UT). 48 hours after transfection cells were treated with 20 nM rapamycin for a further 24 hours. Western blotting was then used to assess protein levels of SRSF1, Mcl-1_L and GAPDH. (<b>D</b>) mir-29b levels measured by real-time PCR in JAR and MCF-7 cell lines (mean (n = 3) ± SEM), ** P≤0.01. Knockdown of SRSF1 in MCF-7 cells (<b>E</b>) and JAR cells (<b>F</b>) was assessed by semi-quantitative RT-PCR in SRSF1 (1) and negative control (NC) siRNA (upper panels). Levels of mir-29b were measured by real-time PCR in SRSF1 (1) and negative control (NC) siRNA treated MCF-7 cells from (E) and JAR cells from (F) along with GAPDH (G) siRNA, vehicle (lipid) only (V), and untreated (UT) cells (lower panels) (mean (n = 3) ± SEM).