Supplementary Figure 7: Inhibition of SETDB1 by Mithramycin A reduces colony formation in vitro and tumor growth in vivo.

a, A schematic illustration of the proposed model for the molecular mechanism underlying the tight regulation of Akt kinase activity and oncogenic functions by the synergy of SETDB1 and PI3K signaling axis. Where indicated, SETDB1 inhibitors were highlighted to combat tumors by reprogramming epigenome and repressing Akt oncogenic signaling. b, IB analysis of GST pull-down and WCL derived from DLD1 cells transfected with indicated constructs treated with different doses of Mithramycin A for 72 hrs before harvest. c-e, IB analysis of WCL derived from DLD1-AKT1/2^-/- infected with WT or K140/142R-Akt1 lentivirus post treatment of different doses of Mithramycin A for 72 hrs (c). The resulting cells were subjected to colony formation assays (d). The experiment was performed twice independently with three repeats, and exhibit similar results (d). Representative images were shown in (d) and relative colony numbers derived from two independent experiments were plotted in (e). (e). f-i, Mithramycin A treatment retards in vivo tumorigenesis of xenografted DLD1 cells. When the tumors of xenografted DLD1 cells reached 100 mm³, the mice were treated with Mithramycin A (0.2 mg/kg) or PBS (as a negative control). Tumor sizes were monitored (f,g) and tumor mass were weighed and presented (h,i). Error bars are mean ± s.e.m, n = 8 mice. P value were calculated using two-way ANOVA analysis (g) or two-tailed unpaired Student’s t test (i). j, IB analysis of tumor lysates derived from the mice bearing A375 xenografted tumors treated with Mithramycin A or PBS. k, A schematic illustration of the proposed model for the molecular mechanisms underlying the tight regulation of Akt kinase activity and oncogenic functions by the SETDB1-KDM4B signaling axis. Where indicated, SETDB1 distributes both in nucleus and cytoplasm to repress gene expressions and promote oncogenic functions by methylating histone substrate H3K9 and non-histone substrate Akt, respectively. Conversely, KDM4B could de-methylate H3K9, Akt-K140/142 and other substrates to integratedly exert its physiological or pathological functions. Detailed statistical tests were described in Methods. Statistical source data for e,g and i 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.