Figure 6

Effects of RasGEF1b overexpression or knock-down on SerpinB2 promoter transcriptional activation. (a) RasGEF1b increases the activity of the SerpinB2 promoter. HEK293 cells were transfected with an empty vector or an expression construct encoding FLAG-RasGEF1b (NM_145839), along with pGLmP-539 SerpinB2 promoter-luciferase reporter and pRL-TK reporter plasmids. Cell lysates were collected after 24 h and assayed for luciferase activity. Transfection experiments were performed ≥ 4 times in independent biological experiments. Data represent the mean ± SEM. Statistical significance was determined for RasGEF1b encoding plasmid compared to pFLAG-CMV4 vector by Student t-test (*p < 0.05). (b) RT-qPCR analysis showing knock-down of RasGEF1b mRNA in shRNA-expressing RAW264.7 cells. Gene expression levels were normalized to the reference gene Rpl32 and calibrated to control shRNA-expressing cells (n = 3 independent experiments). (c, d) Luciferase assays showing SerpinB2 transcriptional activation in puromycin-resistant shRNA control or shRNA RasGEF1b-expressing RAW264.7 cells. Transfections were performed in 24-well plates containing 400 ng and 100 ng of pGLmP-539 and pRL-TK per well, respectively. Twenty-four hours post-transfection, cells were left untreated or treated with LPS 100 ng/ml. Cell lysates were collected after 18 h and assayed for luciferase activity (n = three and two independent experiments for c and d, respectively). Bars represent mean ± SEM. * and ** indicate statistical significance as p < 0.05 and p < 0.01, respectively.