PML-RARα induces a decrease of C/EBP activity as a result of decreased RNA and protein expression. (A) C/EBPα activity in myeloid cells with and without expression of PML-RARα. U937PR9 cells and U937 cells stably transfected with the empty vector (U937MT) or a vector expressing PML-RARα (U937PR9) were pretreated 16 hours with 100μM Zn²⁺SO₄ (Zn) to induce PML-RARα expression. Then,15 million cells were electroporated with a vector driving expression of luciferase cloned in front of 4 C/EBP sites from the G-CSF receptor promoter²⁵  together with a CMV-Renilla vector driving expression of Renilla luciferase.²⁶  Cells were lysed 7 hours after electroporation. Firefly luciferase activity was normalized to Renilla luciferase activity (RLU). *P < .001. (B) Western blot analysis demonstrates a selective decrease in C/EBPα protein after induction of PML-RARα. U937PR9 cells were treated with 100μM Zn²⁺SO₄ (Zn) for the indicated time. Cells were lysed at the indicated time in radioimmunoprecipitation assay buffer and separated onto 10% acrylamide-bis acrylamide gel. After transfer onto a PVDF membrane, the immmunoblot was incubated with an anti-RARα antibody and anti-C/EBPα, β, and ϵ antibodies. Loading control was assessed with an antibody against β-actin. (C) C/EBPα RNA decreases over several days after induction of PML-RARα. Northern blot of U937PR9 cell treated with 100μM Zn²⁺SO₄ (Zn). Cells were collected at the indicated times. A total of 8 μg of RNA were electrophoresed and transferred onto a nylon filter. The filter was hybridized with a probe staining C/EBPα mRNA (top panel). GAPDH (bottom panel) was used to assess the equal loading of each sample. Signals were quantified using densitometric analysis of PhosphorImager data. The amount of C/EBPα mRNA was normalized to GAPDH expression and plotted over time according to arbitrary units (A.U.; ratio C/EBPα to GAPDH RNA). (D) C/EBPα protein expression is decreased in human myeloid cells expressing PML-RARα. C/EBPα protein was detected by Western blot analysis (top panel) in myeloid cell lines, which did not express PML-RARα (HL60, U937, and U937PR9 without zinc) and do express PML-RARα (U937PR9 + zinc, NB4, and HT93). Loading was assessed with an antibody against β-tubulin (bottom panel). The expression of C/EBPα was quantified using the ImageJ program (http://rsb.info.nih.gov/ij/) and normalized to the expression of β-tubulin, and plotted on the bar graph below the blot. Relative expression of C/EBPα (C/EBPα expression divided by β-tubulin expression) is represented with black bars for the cell lines that do not express PML-RARα (HL60, U937, and U937PR9 without zinc) and gray bars for the cell lines that do express PML-RARα (U937PR9 + zinc, NB4, and HT93).