Figure 3.

Suppression of p16 restores anti-leukemic effect of EZH2 inhibition. (a) MLL/ENL leukemia cells were transduced with pSIREN-RetroQ-shp16 (shp16) or control vector and cultured in 2.5 μg/ml of puromycin containing medium for 5 days. The total RNAs were isolated and analyzed for p16 by qRT-PCR. N = 2 for each. (b) shp16 transduced leukemia cells were transduced with pSIREN-ZsGreen control or shEZH2. 1 × 10⁴ of sorted ZsGreen positive cells were placed in 1 ml of methocult M3434 (2.5 μg/ml of puromycin containing) and cultured for 5 days. Colony counts for each group are shown. P-value was calculated by unpaired T test. N = 2 for each. (c) Mice were injected intravenously with 1 × 10⁴ of control or shp16 transduced MLL/ENL leukemic cells. Mice were treated with DZNep or DDW as previously shown in Figure1a. N = 7 for each group. (d) Retrovirus encoding p16-IRES-GFP was infected into MLL/ENL leukemia cells. GFP positive cells were sorted and placed in 1 ml of methocult M3434 and cultured for 5 days. Colony counts for each group are shown. P-value was calculated by unpaired T test. N = 5 for each. (e) Mice were intravenously injected with 1 × 10⁴ of p16-IRES-GFP or mock transduced MLL/ENL leukemic cells. N = 7 for each group.