GNAO1 R209C mutation increases its enzymatic activity and promotes cell proliferation and neoplastic transformation. (A) Enzymatic activity analysis of WT GNAO1 and the R209C mutant. A GTPase activity analysis was performed with the GTPase colorimetric assay kit. (B) The simulated crystal structures of WT GNAO1 and the R209C mutant. (C) Effects of ectopic expression of WT GNAO1 or the R209C mutation on cell proliferation in Reh, UoCB6, and Nalm6 cells. Cell proliferation analysis was performed using the Click-iT EdU Assay Kit. (D) Representative images of soft agar colony formation. Scale bar, 1 mm. (E) Quantification of soft agar colony formation in panel D. (F) Representative images of soft agar colony formation of the PBMC specimens from twin B before the onset of leukemia, twin A at the onset of leukemia, or twin A at remission. Data are representative of 3 or 4 independent experiments with similar results. Scale bar, 1 mm. (A,C,E) data are expressed as the mean ± SD. *P < .05; **P < .01, by 2-tailed Student t test.