Figure 2.

Molecular characterization of NUP98-JADE2 and JADE2 in ATRA-mediated responses and myeloid differentiation. (A) HeLa cells were cotransfected with RARE reporter and the indicated pCMV-HA plasmids. EV, empty vector; NJ, NUP98-JADE2; PR, PML-RARA. Luciferase activities were determined as described in supplemental Methods. (B) HeLa cells were cotransfected with RARE reporter and an increasing pCMV-HA-NUP98-JADE2 (NJ), pCMV-HA-JADE2 (J2), or the empty pCMV-HA (EV), and then treated with ATRA or DMSO for 6 hours before analysis. Results are presented as fold induction vs DMSO treatment. (A-B) *P < .05, **P < .01, and ***P < .001 vs EV, respectively by 1-way analysis of variance followed by Dunnett test. (C) NB4 cells transduced with NUP98-JADE2 (NJ) or control (EV) lentiviruses were treated with ATRA or DMSO. CD11b on GFP-positive cells was measured by flow cytometry. *P < .05 and **P < .01, respectively by paired t test. (D) Left, confirmation of JADE2 knockdown in NB4 cells after 24 hours of siRNA transfection by quantitative RT-PCR. After the transfection, cells were treated with 0.1 µM of ATRA or DMSO for 3 days. CD11b was measured by flow cytometry (middle) and CEBPE by quantitative RT-PCR and normalized to GAPDH (right). **P < .01 and ***P < .001 vs the negative siRNA, respectively by 1-way analysis of variance followed by Dunnett test. (E) Quantitative RT-PCR to validate selected JADE2 target genes in NB4 cells after 48 hours of siRNA transfection. **P < .01 vs negative siRNA by the Mann-Whitney U test. (A-E) Results are expressed as mean plus standard error from 3 independent experiments. (F) Pathway analysis of the 56 downregulated genes by ConsensusPathDB.¹⁵ The top 15 enriched Gene Ontology Biological Processes are shown. (G) Expression of the downregulated genes in purified polymorphonuclear cells (PMN) and granulocyte-monocyte progenitors (GMP). Data (from GSE42519) were available for 54 of the downregulated genes. Genes that are upregulated by ≥twofold (log₂ fc≥1) in PMN are boxed.