Figure 2.

Promyelocytic leukemia/retinoic acid receptor α (PML/RARa) inhibits NF-E2 p45-related factor 2 (NRF2) transcriptional activity by preventing its binding to antioxidant response elements (ARE) motifs. (a) mRNA expression of NRF2 target genes: HMOX1, NQO-1 and AKR1C-1 in 13 APL, 12 AML and 5 normal bone marrow (NBM) samples. * p ≤ 0.05; **: p ≤ 0.01; ***: p ≤ 0.001 by the Mann–Whitney test, not normal distribution. (b) NRF2 mRNA expression induction in a 24 h time course after ZnSO4 addition is higher in PR9 than in Mock control cells. The experiments were performed in triplicate. (c) NRF2 protein level increases and persists higher in a 24 h time course after treatment with ZnSO₄ in Mock control cells; conversely, in PR9 cells after a short lived augment, it abates due to the presence of PML/RARa. The experiments were done by triplicate. **: p ≤ 0.005 by unpaired t-test. (d) PML/RARa expression after treatment with ZnSO₄ in PR9 cells inhibits the transcriptional activity of NRF2 as measured by HMOX1 expression. The experiments were done by triplicate. **: p ≤ 0.005 by unpaired t-test. (e) The squares indicate the position of putative ARE motifs relative to the HMOX1 transcription start site (TSS). The arrows indicate the position of primers used to analyze the putative ARE binding site in the HMOX1 gene. The experiment were done by triplicated. *: p ≤0.05 by unpaired t-test. (f) PML/RARa decreases binding of NRF2 to the HMOX1 promoter region. The binding of NRF2 markers to DNA was measured by a quantitative ChIP assay in PR9 and Mock cells after treatment with ZnSO₄ (100 µM). Data are shown as fold-enrichment of ChIP DNA versus input DNA. GAPDH was used as negative control. Data are representative of four independent experiments. p = 0.02 by Mann–Whitney test. Not normal distribution.