Extended Data Fig. 3. The loss of tissue-specific Hsd3b6 activity of conditional Hsd3b6^ΔMG mice causes atrophy of the meibomian gland.

a, Immunohistological validation of Hsd3b6 protein expression in the meibomian gland and adrenal gland of WT (Hsd3b6^fx/fx) and Hsd3b6^ΔMG (Hsd3b6^fx/fx;K14-Cre) mice. ZG, zona glomerulosa. ZF, zona fasciculata. Scale bars, 100 µm. Data are representative of three independent mice with similar results. b, The mRNA levels of Hsd3b6 and Hsd3b1 in the meibomian gland and adrenal gland of WT and Hsd3b6^ΔMG mice (n = 2 biologically independent samples, for each tissue). n.d., not detectable. c, The mRNA levels of Hsd3b6 and Hsd3b1 in the skin, testis, and ovary of WT and Hsd3b6^ΔMG mice (n = 2 biologically independent samples, for each tissue). Male mice were used for analysis except for the ovary. Values were determined by qRT-PCR and normalized to those of ribosomal protein large P0 gene. n.d., not detectable. d, Chromatograms showing the loss of local 3β-HSD activity in the meibomian gland of Hsd3b6^ΔMG mice. 3β-HSD enzymatic activities in WT and Hsd3b6^ΔMG eyelids were determined by measuring the conversion of ³H-DHEA (green) to ³H-androstendione (magenta). e, Representative images of whole-mount meibomian gland staining of upper and lower eyelids of WT and Hsd3b6^ΔMG mice. mo, months old. Arrow indicates the area of gland dropout. Scale bars, 1 mm. f, Quantification of the area of the meibomian glands of WT and Hsd3b6^ΔMG mice (n = 9 mice for each genotype, 3 mo). ^***P < 0.0001, unpaired two-sided t-test. All values are presented as mean ± SEM.