FIG 7.

CITED2 curbs HIF1α protein level and function by preserving EGLN3 expression. (A) BMDMs from Lyz2^cre and Cited2^fl/fl:Lyz2^cre mice were stimulated with 100 ng/ml of LPS for 6 h. Total RNA from the cells was analyzed for expression of positive and negative regulators of Hif1α at the mRNA level by quantitative PCR. Untreated Lyz2^cre BMDMs were used as a control, and relative fold change over control is indicated (n = 4). (B to G) Primary macrophages from Lyz2^cre and Cited2^fl/fl:Lyz2^cre mice were stimulated with 100 ng/ml of LPS for 6 h and analyzed for EGLN1 (B and C), EGLN2 (D and E), and EGLN3 (F and G) mRNA and protein expression by quantitative-PCR and Western blot analyses (n = 4). (H to K) RAW 264.7 cells were transfected with Cited2-specific siRNA and were further cotransfected with the Egln3 plasmid. The cells were stimulated with 100 ng/ml of LPS for 6 h. (H) Protein extracts from these experiments were evaluated for HIF1α expression by Western blotting. (I to K) Total RNA from these experiments was analyzed for Il1α, Il1β, and Adm mRNA expression by quantitative PCR (n = 4). The beta-actin and 36B4 genes were used as housekeeping genes for Western blot and quantitative-PCR analyses, respectively. Data were analyzed either by Student's t test (A, B, D, and F) or by 2-way ANOVA (I to K). N.S., not significant; *, P < 0.01; **, P < 0.0001. All values are reported as means and SD.