Regulatory Effects of Sestrin 3 (SESN3) in BCR-ABL Expressing Cells
Figure 5
Inhibitory effects of SESN3 but not SESN2 on primitive BCR-ABL expressing leukemic progenitors.
A. KT-1 cells were transiently nucleofected with either empty vector (E.V.) or a SESN3 expressing plasmid. Levels of SESN3 were quantified at 24 hours post-nucleofection by immunoblotting. B. KT-1 cells transiently nucleofected with either empty vector (E.V.) or SESN3 were plated in methylcellulose 24 hours post-nucleofection. Leukemic CFU-L colonies were allowed to develop in clonogenic assays in methylcellulose and scored on day 6. Data are expressed as percentage of control untreated colonies and represent means ± S.E. of 5 independent experiments. C. KT-1 cells were transiently nucleofected with either empty vector (E.V.) or SESN2 expressing plasmid. Levels of SESN2 were quantified at 24 hours post-nucleofection by immunoblotting. D. KT-1 cells transiently nucleofected with either empty vector (E.V.) or SESN2 expressing plasmid were incubated in clonogenic assays in methylcellulose. Leukemic CFU-L colonies were scored on day 6 and data are expressed as percentage of control untreated colonies and represent means ± S.E. of 4 independent experiments. E. BV173R cells were transiently nucleofected with either empty vector (E.V.) or SESN2 or SESN3 expressing plasmid. 48 hours post-transfection, equal number of cells were plated and allowed to proliferate for 120 hours. Proliferation was measured by WST-1 assay at the indicated times. Data are expressed as the absorbance at 450 nm and represent means ± S.E. from 3 independent experiments, * p = 0.0018 comparing SESN3 nucleofected cells vs. E.V. nucleofected cells on day 4, ** p = 0.0068 comparing SESN3 nucleofected cells vs. E.V. nucleofected on day 5.