Figure 4. Targeting of NSD3 or CHD8 leads to differentiation of AML cells and suppression of BRD4-dependent gene expression.

(A–B) (top) Competition-based assays to evaluate the effect of NSD3 or CHD8 LMN shRNAs on RN2 cell proliferation. GFP percentages are normalized to d2 measurements. All error bars represent the SEM of three independent biological replicates, (bottom) Western blotting analysis of whole cell lysates prepared from RN2 cells transduced with the indicated TRMPV-Neo constructs following 48 hours of dox treatment. A representative experiment of three biological replicates is shown. (C–D) Flow cytometry analysis of c-kit and Mac-1 stained RN2 cells following TRMPV-Neo shRNA induction with dox for 96 hours. Gating was performed on dsRed+/shRNA+ cells. A representative experiment of three biological replicates is shown. (E–F) Light microscopy of May-Grünwald/Giemsa-stained RN2 cells expressing the indicated NSD3 shRNAs or Chd8 sgRNAs, in the presence or absence of ectopic c-Myc expression. For sgRNA experiments, an RN2 line stably expressing Cas9 was used. shRNA expression was induced using the TRMPV-Neo vector treated with dox for 4 days. For F, cells were imaged 6 days following transduction with the indicated LRG sgRNAs. Imaging was performed with a 40x objective. A representative image of three independent biological replicates is shown. (G–H) Gene Set Enrichment Analysis (GSEA) of RNA-seq data obtained from RN2 cells expressing NSD3 TRMPV-Neo shRNAs (induced with dox for 48 hours) or RN2-Cas9 cells expressing Chd8 LRG sgRNAs (4 days following transduction). Two independent NSD3 shRNAs or Chd8 sgRNAs were compared to a Ren.713 shRNA or Rosa26 sgRNA, respectively in this analysis. NES: Normalized enrichment score. For each of the indicated gene sets shown, the false discovery rate and nominal p-value were <0.01. See also Figure S4 and Table S3.