Figure 2. PML-RARα inhibits the transcription of DAPK2 in APL cells.

(A) Schematic representation of putative RAREs in the proximal DAPK2 promoter. (B) ChIP assays using NB4 cells. Histone 3 (His) and IgG served as positive and negative controls, respectively. PCR was performed using primers encompassing the −867 or the −733 PML-RARα-binding site in the DAPK2 promoter and an unrelated sequence 1.6 kb downstream of the GC box as a negative control. (C) Confirmation of ectopic PML-RARα expression. PML-RARα RNA levels were evaluated using qPCR. mRNA levels were normalized to the HMBS housekeeping gene. Results are shown as n-fold regulation compared with NB4 APL cells. Und., Undetectable. (D) Western blot analysis of DAPK2 and PML-RARα expression in U937 PML-RARα cells. Total protein was extracted and submitted to immunoblotting using anti-DAPK2 and anti-RARα. GAPDH is shown as a loading control. (E) Inhibiting PML-RARα increases DAPK2 expression. NB4 cells were transduced with shRNA targeting PML, and PML-RARα knockdown efficiency was confirmed at the mRNA level. DAPK2 mRNA levels were measured by qPCR. DAPK2 expression was normalized to the HMBS housekeeping gene. Results are shown as n-fold regulation compared with control-transduced NB4 SHC002 cells. (F) Western blot analysis of DAPK2 and PML-RARα expression in NB4 PML knockdown cells. MWU: *P < 0.05.