Figure 6.

Knockdown of C/EBPβ reduces Bt₂cAMP-induced aromatase expression and enzyme activity. LSMCs were transfected without oligonucleotide (mock), with a nontargeting, control siRNA or C/EBPβ siRNA for 36 h, then starved for 12 h, followed by treatment with or without Bt₂cAMP for 48 h. A, Immunoblotting for C/EBPβ was performed to determine the knockdown efficiency by C/EBPβ siRNA. Equal loading was confirmed by immunoblotting with anti-actin antibody. Both C/EBPβ-1 (45 kDa) and C/EBPβ-2 (42 kDa) were detected using the anti-C/EBPβ antibodies: C/EBPβ-LAP and C/EBPβ phosphorylated at Thr²³⁵ [P-C/EBPβ (Thr²³⁵)]. B, Aromatase mRNA expression levels after transfection of C/EBPβ siRNA were compared with wild type (without transfection), mock (transfection reagent only), and control siRNA (nontargeting control siRNA) treated with Bt₂cAMP (black bars) or vehicle (white bars). Aromatase mRNA expression levels were quantified by real-time RT-PCR and normalized to GAPDH mRNA. Data are represented as the average + sem from triplicate experiments. Transcripts from only vehicle, untransfected cells were arbitrarily assigned a unit of one, against which expression from transfected cells was compared. *, P < 0.05 (ANOVA). C, Aromatase enzyme activity was compared as in B using a [³H]water releasing assay. Data are represented as the average + sem from triplicate experiments. *, P < 0.05 (ANOVA). All of the results were reproducible in at least three subjects.