Supplementary Figure 4: Depletion of SETDB1 decreases Akt oncogenic functions.

a-e, IB assays of WCL derived from OVCAR5 (a) and DLD1 (b) cells infected with shRNAs against SETDB1. Resulting cells were subjected for colony formation and soft agar assays (c-e, top panel). The experiment was performed twice independently with three repeats, and exhibit similar results (c-e). Representative images were shown in (c-e, top panel) and relative colony numbers derived from two independent experiments were plotted in (c-e, bottom panel). f, The glucose uptake was measured with FACS as mentioned in Material section. The experiment was performed twice independently with three repeats, and exhibit similar results (f). Relative glucose levels derived from two independent experiments were plotted in f. g,h, IB analysis of WCL derived from A375 cells infected with lentivirus against SETDB1, and sequentially infected with retrovirus encoding myr-Akt1 (g). Resulting cells were subjected to colony formation assays (h, bottom panel). The experiment was performed twice independently with three repeats, and exhibit similar results (h). Representative images were shown in (h, top panel) and relative colony numbers derived from two independent experiments were plotted in (h, bottom panel). i-k, IB analysis of HA- or Akt1-IP as well as WCL derived from A375 or HEK293 cells transfected with the indicated constructs. Resulting cells were serum-starved for 18 hrs and stimulated with IGF (30 ng/ml) (i,k) or insulin (0.03 μM) (j) in different time points before harvest for IB analysis. l,m, IB analysis of IP products and WCL derived from HEK293 cells transfected with the indicated constructs treated with (l) or without (m) PI3K inhibitors. Source data for c, d, e, f and h 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.