CRA-induced HO-1 expression in DCs. (A) BM-DCs were treated with CRA (10 μM) in the presence of vehicle alone or LPS (10 ng/mL) for 6 hours. Total RNA was isolated from these cells, and subjected to real-time PCR analysis for mRNA expression for HO-1 and GAPDH (mean ± SD, n = 3). Data are normalized to GAPDH mRNA expression as mentioned in “Measurement of in vitro impacts of CRA on LPS-induced DC maturation.” (B-C) BM-DCs were cultured with CRA in the presence of vehicle alone or LPS to examine time kinetics (10 μM; B) and dose dependency (6 hours) for HO-1 protein expression (C). The whole-cell extracts were assessed by Western blotting for HO-1 protein expression. The whole-cell extracts were also subjected to Coomassie blue staining. (D) HO-1 enzymatic activity was examined in whole-cell extracts from BM-DCs treated with CRA or vehicle alone in the presence or absence of LPS for 6 hours. Data are shown as nmol of bilirubin/minute per mg protein (mean ± SD, n = 3). (E-F) CD3⁺ T cells freshly purified from C57BL/6 mice were treated with CRA (10 μM) or CoPPIX (50 μM) in the presence (F) or absence (E) of PMA plus ionomycin for 6 hours and then examined for HO-1 mRNA expression by real-time PCR. Data are normalized to GAPDH mRNA expression (mean ± SD, n = 3). Asterisks indicate statistically significant (**P < .01) differences compared with the nontreated control samples.