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. 2019 Dec 30;12(1):95. doi: 10.3390/cancers12010095

Figure 3.

Figure 3

Promyelocytic leukemia/retinoic acid receptor α (PML/RARa) binds to NF-E2 p45-related factor 2 (NRF2), promotes its cytoplasmic degradation and shortens its lifespan. (a) At confocal microscopy NRF2 translocates to the cytoplasm eight hours following PML/RARa expression induction. The arrow indicates promyelocytic leukemia-nuclear bodies (PML-NBs). (b) Cytoplasmic translocation is confirmed by nuclear/cytoplasmic fractionation. (c) PR9 cells where treated with ZnSO4 100 μM for four hours, after that cross-immunoprecipitation experiments where done showing that PML/RARa interacts with NRF2. (d) Co-immunoprecipitation of NRF2 in the cytoplasm and nucleus using PR9 cells previously treated with ZnSO4 for four hours demonstrated an interaction between PML/RARa and NRF2 in both fractions. (e) Hek293T cells were transfected with pS5G or pS5G-PML/RARa, after 24 h where treated with ZnSO4 100 μM to evaluate the effect of PML/RARa on NRF2 induction. PML/RARa inhibit the expression of NRF2 (f) PR9 cells and the control Mock cells were treated with ZnSO4 100 μM for two hours and then with cycloheximide (CHX) 100 μg/mL for five hours. Time 0 refers to the moment in which cycloheximide was added, +2 h after zinc induction. PML/RARa reduce significantly the half-life of NRF2. (g) HL60 cells were transfected with pS5G or pS5G-PML/RARa for 24 h, then treated with ZnSO4 100 μM for two hours and then with CHX 100 μg/mL for four hours. PML/RARa reduce the half life of NRF2. This experiment was done twice. All the other experiments were done by triplicate. *: p ≤ 0.05; **: p ≤ 0.01 by unpaired t-test.