Figure 4. Staphylococcus epidermidis but not Corynebacterium singulare expresses urocanase. S epidermidis and C singulare strains were cultured in liquid Luria Broth medium for 24 h. In some cultures, 250 μg cis-UCA or 250 μg trans-UCA were added. At the end of the culture experiment, the presence for urocanase (UROC1) or control GAPDH protein was quantified by western blot. (a) Results depict representative immunoblots and means ± SD and individual protein expression data normalized to GAPDH expression level in (b) 24-h cultures. P-values were determined using the 2-way ANOVA. h, hour; UCA, urocanic acid.
Supplementary Figure S1. A large proportion of trans-UCA present on the skin surface converts to cis-UCA directly after UVB exposure. The shaved dorsal skin of C57BL/6 mice was exposed to 618 mJ/cm2 UVB, and approximately 2 min after exposure, skin samples were collected by tape stripping (a). trans- (b) and cis-UCA (c) detected in skin samples were detected and quantified by HPLC-MS method. Graphs depict the means ± SD and individual quantitative data for each isomer. Respective cis-/trans-UCA ratios in each sample are also shown (d). Results were pooled from 3 independent experiments with 8–9 mice per group. HPLC-MS, high-performance liquid chromatography-mass spectrometry; min, minute; UCA, urocanic acid.
Supplementary Figure S5. cis-UCA induces dose-dependent suppression of contact hypersensitivity to DNFB. C57BL/6 mice were pretreated on shaved dorsal skin with graded doses (125, 250, and 500 μg) of cis-UCA, with trans-UCA (500 μg), UVB (618 mJ/cm2), a vehicle (70% EtOH), or left untreated. (a) Three days later, mice were sensitized by applying 0.5% DNFB (25 μl) on the shaved abdomen (except those from the “irritation control” group), and the ears were challenged next with 0.13% DNFB (2 × 5 μl) 5 days later. (b) Results depict the means ± SD and individual ear swelling data, as measured 24 h after the DNFB challenge. The percentage of immunosuppression induced in cis-UCA– versus vehicle-treated groups is also shown. Data are pooled from 2 different experiments with 5 mice per group. P-values were determined using Dunnett's test for multiple comparisons. DNFB, 2,4-dinitrofluorobenzene; EtOH, ethanol; ns, not significant; UCA, urocanic acid.
