DN effects of dnPPARγ-Flag fusion protein on inflammatory molecule genes. (A) DNA binding of dnPPARγ-Flag fusion protein on promoter regions of Api6, IL-1β, IL-6, MMP12, and TNF-α genes using ChIP assay followed by real-time PCR (n = 4). Relative DNA-binding levels were determined by 2^(−ΔΔCt), in which ΔΔCt = ΔCt _(+DOX) − ΔCt _(−DOX). Testing samples Ct value was normalized by each input DNA fraction Ct value. Therefore, ΔCt = Ct _(testing) − (Ct _(input) − Log₂ (input dilution factor). Values are means ± SD. *P < .05; **P < .01. (B) Real-time PCR analysis of mRNA expression levels of inflammatory cytokines in BM MDSCs from c-fms-rtTA/(tetO)₇-CMV-dnPPARγ–bitransgenic mice that were treated or untreated with doxycycline for 3 months (n = 5). The mRNA expression level of GAPDH was used for normalization. ΔCt = Ct_(testing) − Ct_(GAPDH). Relative expression levels were determined by 2^(−ΔΔCt), in which ΔΔCt = ΔCt (+DOX) − ΔCt _(−DOX). Values are means ± SD. **P < .01. (C) Protein expression levels of IL-1β, IL-6, and TNF-α in serum and bronchoalveolar lavage fluid from the lungs of 3-month doxycycline-treated or untreated bitransgenic mice as shown by ELISA (n = 5). Values are means ± SD. *P < .05. (D) Percentage of CD11b⁺GR-1⁺ cells in 3-month doxycycline-treated or untreated bitransgenic mice after injection of anti–IL-1β, anti–IL-6, and anti–TNF-α Abs in neutralization studies. −DOX indicates doxycycline-untreated bitransgenic mice; +DOX, doxycycline-treated bitransgenic mice. *P < .05, **P < .01.