Supplementary Figure 2: Depletion of SETDB1 decreases Akt kinase activity.

a, A sequence alignment of nuclear acids between WT and K140/142R mutant form of Akt1 indicates the strategy for generating the Akt1-K140/142R knock-in (KI) mice. As indicated, TGG sequence in WT-Akt1 was selected as the sgRNA PAM sequence. b,c, The gel image illustrates the Sac II-digesting genomic DNAs derived from litters selected from knock-in mice, and the corresponding DNAs were subjected for sequencing (c). d-f, Frequency of genotypes produced from Akt1 KI/WT mouse intercrosses (d). Numbers in parentheses indicate the expected number by Mendelian ratio. Growth of Akt1^WT versus Akt1^KI/KI female and male mice. Data were shown as mean+/-s.e.m. (male, n = 26 mice in Akt1^WT group and n = 18 mice in Akt1^KI/KI group; female, n = 19 mice in Akt1^WT group and n = 14 mice in Akt1^KI/KI group). g,h, The mice derived from the same litter were imaged at age of 4 weeks old, and were euthanized and their organs were dissected (g) and weighed, and further subjected to H&E and IHC staining (h). Scale bar, 50 μm. The experiment in h was performed twice, independently, with similar results. i, IB analysis of IP and WCL derived from HEK293 cells transfected with indicated constructs. j, Genomic alterations of SETDB1 are mutually exclusive with the alterations of the PI3K/Akt pathway-related genes (PTEN, EGFR, PIK3CA and AKT1) from the TCGA database including melanoma and breast cancer. k, A schematic illustration of the potential regulation of Akt1 with SETDB1 or EZH2, respectively. Where indicated, SETDB1 is the upstream regulator of Akt capable of methylating Akt, whereas EZH2 has been reported to be the downstream substrate of Akt, which can be phosphorylated by Akt. l,m, IB analysis of GST pulldown and WCL derived from HEK293 cells transfected with indicated constructs. Statistical source data for e and f are shown in Supplementary Table 2. All Western-blots above were performed twice independently with similar results. Scanned images of unprocessed blots are shown in Supplementary Fig. 8.