Fig. 6: Absence of R391 methylation attenuates AKT activation and tumor growth.

a, b IB analysis of WCLs derived from DLD-1-AKT1/2^−/− cells expressing AKT1-WT or R391K mutant. Where indicated, cells were serum-starved for 16 h and then treated with 100 nM insulin for 0, 30, and 60 min before collection. c In vitro kinase assays using immunopurified HA-AKT1-WT or R391K as the kinase source and recombinant GST-GSK-3β purified from bacteria as the substrate. d DLD-1-AKT1/2^−/− cells expressing AKT1-WT or R391K mutant were subjected to cell proliferation assays. Data are shown as the mean ± SD of n = 3 independent experiments. e Representative images of colony formation assays. f Quantification of colonies. Data are shown as the mean ± SD of n = 3 independent experiments. g–i DLD-1-AKT1/2^−/− cells expressing AKT1-WT or R391K mutant were subjected to mouse xenograft assays (injection both sides of mice). Tumor growth were monitored (g) and dissected tumors were weighed (h, i). Data are shown as the mean ± SEM g or mean ± SD (i) of n = 5 mice (WT) or 4 mice (R391K). j Representative images of TUNEL assays in xenograft tumors. Scale bar, 50 μm. k Quantification of TUNEL-positive cells in xenograft tumors. Data are shown as the mean ± SD of n = 4 tumors. l Representative images of IHC staining of Ki-67 in n = 4 xenograft tumors. Scale bar, 100 μm. Statistical significance was determined by two-tailed Student’s t-test in f, i, k and by two-way ANOVA in d, g. Similar results were obtained in n ≥ 2 independent experiments in a–c. Uncropped immunoblots and statistical source data are provided in Source Data files.