R26(EF1α-tTA/TetO-mCh-Rs1) function in mouse ES cells. (A) FACS analysis showing doxycycline-inducible mCherry expression in E14 mouse cells carrying the Exp-R26(EF1α-tTA/TetO-mCh-Rs1) construct. (B) Induction of Rs1 expression and treatment with the agonist RS67333 results in increased cAMP accumulation in mouse ES cells. Both basal and ligand-induced increases in cAMP are detectable. In addition, serotonin does not induce increased cAMP accumulation in either the wildtype or Rs1-expressing cells. (C) Schematic showing the differentiation protocol for making suspension EBs. (D) Expression and ligand activation of Rs1 during EB formation results in larger EB size. (E) Quantitation of EB size in the different culture conditions using ImageJ. A minimum of 114 EBs were measured for each condition. The analysis was performed on three separate EB differentiation experiments with similar results. Error bars represent average +/- 1 SD.