Fig. 10. Effects of WT and mutant Rab5 and dynamin2 on LPA₁ stimulation of SRF-mediated transcription. (A) HepG2 cells were transiently transfected in serum-free medium with plasmids encoding SRE-luciferase, pRL-TK alone or with an expression vector for FLAG-tagged LPA₁. Cells were incubated in the absence or presence of 1 µM LPA for 16 hours in serum-free medium prior to determination of luciferase activity (see Materials and Methods). Normalized luciferase activity is first calculated as the ratio of SRE-encoded firefly luciferase activity to TK-encoded Renilla luciferase activity. Next, the activities of all the samples are expressed as a fraction of the data collected from cells expressing LPA₁ and SRE-luciferase, which had not been treated with LPA. Note that, in the absence of LPA₁ expression, LPA treatment does not induce the SRE-luciferase construct. The data are expressed as the mean ± s.e.m. from two independent experiments that were performed in triplicate. (B) HepG2 cells were transiently transfected with plasmids encoding SRE-luciferase, pRL-TK and FLAG-LPA₁ alone or with either GFP-tagged WT or GFP-tagged mutant Rab5 or Dyn2. The data represent the means ± s.e.m. of three measurements from a representative experiment that was repeated three times. (C) The data shown in B above was re-analyzed to determine the fold induction of luciferase activity by LPA treatment. Fold induction was calculated by first dividing the normalized luciferase data from each LPA-treated sample by the luciferase data of the corresponding untreated samples and then averaging these ratios. The data shown is the average fold induction ratios ± s.e.m. *P<0.05 compared with LPA₁ alone.