(A) Negative selection shRNA screen targeting chromatin regulators in murine B cell acute lymphoblastic leukemia (B-ALL). shRNAs are rank ordered by the fold-change in GFP positivity over 10 days in culture, which represents a competition-based assay in which loss of GFP positivity reflects shRNA-postive cells becoming outcompeted by shRNA-negative cells. (B–E) Competition-based assays and Western blotting to evaluate the effect of TRIM33 shRNAs on B-ALL, 38B9, acute myeloid leukemia (AML), or T-cell acute lymphoblastic leukemia (T-ALL) cells. GFP percentages are normalized to day 2 measurements. Results are the average of three biological replicates. (F) Annexin V/DAPI staining following transduction of B-ALL cells with the indicated MLS shRNAs on day 3 post-transduction. Representative experiment of three biological replicates is shown. (G) shRNA transgenic mouse strategy. TRE: tet(doxycycline) response element; rtTA-M2: reverse tet transactivator M2 variant (tet-on). (H) Western blotting performed of indicated tissue lysates prepared from mice treated with dox for 4 weeks. Representative experiment of three biological replicates is shown. (I–J) Flow cytometry analysis using the indicated antibody stainings of whole bone marrow or spleen. B220 and Cd19: B lymphoid, Ter119: erythroid, Gr1 and Mac1: myeloid, Cd3: T lymphoid. Gating was performed on GFP+/shRNA+ cells prior to quantifying marker positivity. The GFP+ percentage in bone marrow was ∼75% and in the spleen was ∼15%. Mice were administered dox for 1 week or 4 weeks, with both timepoints giving similar results. Results shown are the average 4 or 5 mice. All error bars in this figure represent S.E.M.