Fig. 3. Circadian 3β-HSD enzymatic activity and age-related decline of its coenzyme NAD⁺ in the meibomian gland.

a, Organotypic eyelid slice from Per2^Luc/+ mice and magnified images of PER2::LUC luminescence with Nile red staining. Scale bars, 100 μm. b, Circadian oscillation of PER2::LUC. Arrows indicate representative rhythmic, basal cell layers. Bmal1^−/−;Per2^Luc/+ eyelid slice failed to keep oscillation. c, Dual immunolabelling of PER2::LUC and Hsd3b6. Arrows indicate representative double-labeled cells. Scale bar, 50 μm. d, Time-of-day-dependent changes in 3β-HSD activity in WT and Bmal1^−/− meibomian gland (n = 4 mice per time point). e, Quantification of meibomian gland area in WT (n = 4) and Bmal1^−/− (n = 5) mice. ^*P = 0.0229. f, 24 h mRNA expression of Per2 and Hsd3b6 in laser-microdissected meibomian gland (n = 4 mice per data point). Scale bar, 200 µm. g, 24 h temporal variations in Hsd3b6 protein expression. Immunoreactivities were abolished in Hsd3b6^−/− mice. Requirement of NAD⁺ for dehydrogenase activity of Hsd3b6 (bottom). IB, immunoblot. h, NAD⁺ levels in the meibomian gland at ZT03 and ZT15. n = 17, ZT03; n = 14, ZT15; biologically independent laser-microdissected tissue samples. ^*P = 0.0477. i, Meibomian gland NAD⁺ levels in WT (n = 8) and Bmal1^−/− (n = 7) mice. ^*P = 0.0261. j, Relative mRNA levels of Hsd3b6 in laser-microdissected meibomian gland, normalized to those of Itgav in WT and Bmal1^−/− mice. n = 3 mice per group. k, Meibomian NAD⁺ levels at 8, 14, 20 and 24 mo. n = 8 WT mice per group except n = 7 for 24 mo. ^*P = 0.0301; ^**P = 0.0011, versus 14 mo. l, Hsd3b6 mRNA levels at 2–3, 19–20 and 24–25 months of age. n = 8, 2–3 mo; n = 4, 19–20 mo; n = 4, 24–25 mo mouse meibomian glands. Values are presented as mean ± s.e.m. Statistics in k,l were one-way ANOVA and Bonferroni test; statistical test in e,h,i was unpaired, two-sided Student’s t-test. Data presented in a–c,g are representative of four biologically independent experiments with similar results.