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. 2018 Feb 12;38(5):e00452-17. doi: 10.1128/MCB.00452-17

FIG 4.

FIG 4

CITED2 cooperates with PPARγ to enhance IL-4-induced anti-inflammatory gene expression. (A) RAW 264.7 cells were transfected with a PPAR luciferase reporter construct in the presence of pCMV6 or pCMV6-Cited2 plasmid. The cells were stimulated with 10 ng/ml IL-4 in the presence of rosiglitazone (RSG) or T0070907, and cell lysates were analyzed for luciferase activity (n = 3). (B) RAW 264.7 cells were transfected with a PPAR luciferase reporter construct in the presence of control or Cited2-specific siRNA. The cells were stimulated with 10 ng/ml IL-4 in the presence of 10 μM RSG, and cell lysates were analyzed for luciferase activity (n = 3). The efficiency of siCited2 siRNA was confirmed by Western blot analysis. (C) RAW 264.7 cells were transfected with either pCMV6 or pCMV6-Cited2 plasmid. The cells were stimulated with 10 ng/ml of IL-4 for 18 h, and mRNA expression of PPARγ was analyzed by quantitative PCR (n = 4). (D and E) BMDMs from Lyz2cre and Cited2fl/f:Lyz2cre mice were stimulated with 10 ng/ml of IL-4 for 18 h. PPARγ mRNA and protein expression was evaluated by quantitative-PCR and Western blot analyses, respectively (n = 4). (F) BMDMs from wild-type mice were stimulated with 10 ng/ml of IL-4 for 1 h. The cell lysates were immunoprecipitated with anti-PPARγ antibody and immunoblotted with the anti-CITED2 antibody (n = 3). (G) Lyz2cre and Cited2fl/fl:Lyz2cre BMDMs were stimulated with IL-4, and ChIP analysis was performed on the Arg1 enhancer and Retnla promoter utilizing anti-PPARγ and anti-CITED2 antibodies (n = 3). (H to K) BMDMs from Lyz2cre and Cited2fl/fl:Lyz2cre mice were stimulated with 10 ng/ml IL-4 in the presence or absence of RSG. Total RNA was isolated, and mRNA expression of Arg1, Retnla, Lpl, and Tgm2 were analyzed by quantitative PCR (n = 3). The beta-actin gene was used as a housekeeping gene for Western blotting, and the 36B4 gene was used as a housekeeping gene for quantitative-PCR analysis. Each experiment was performed at least three times with three replicates. Data were analyzed by either 2-way ANOVA (A, B, and H to K) or Student's t test (C, D, and G). N.S., not significant; *, P < 0.01; **, P < 0.0001. All values are reported as means and SD.