Figure 3. SETD1A FLOS domain provides a non-redundant role in leukemia cell proliferation.

A. Relative expression level of Setd1a in MLL-AF9 leukemia cells was quantified by qRT-PCR at 4 days post-tamoxifen treatment. This experiment was repeated 3 times with 3 biological replicates in each experiment.

B. MLL-AF9 leukemia cells were transfected with SETD1A mutant constructs. The cells were treated with tamoxifen, then assayed for colony-forming potential. Representative data from one out of 2 independent experiments with 3 biological replicates are shown.

C. The chimeric constructs of human SETD1A and human SETD1B are shown as schematic illustrations.

D. Setd1a^fl/fl;CreER cells were transfected with SETD1 chimeric constructs and relative exogenous gene expression level was quantified by Taqman probes which can detect either human SETD1A or SETD1B. This experiment was repeated 2 times with 3 biological replicates in each experiment.

E. MLL-AF9 leukemia cells were transfected with SETD1 chimeric constructs and monitored for cell number increase within 3 days from day 4 post-tamoxifen treatment. This experiment was repeated 3 times with 3 biological replicates in each experiment.

F. Annexin V⁺DAPI⁻ population in Setd1a^fl/fl;CreER cells expressing SETD1 chimeric constructs was analyzed at day 5 post-tamoxifen treatment. This experiment was repeated 3 times with 3 biological replicates in each experiment.

G. Representative morphology of colonies in Setd1a^fl/fl;CreER cells expressing SETD1 chimeric constructs are shown.

H. Single cell-derived colonies of Setd1a^fl/fl;CreER cells expressing SETD1 chimeric constructs were isolated after tamoxifen-treatment followed by colony-forming assay. Setd1a alleles of 5 clones are genotyped by PCR.