Extended Data Fig. 2: Generation of Hsd3b6^−/− mice and phenotypic analysis of the meibomian gland function with the female mice, related to Fig. 2c-i.

a, Schematic structure of the wild-type (Hsd3b6⁺), targeted (Hsd3b6^fxneo), floxed (Hsd3b6^fx) and deleted (Hsd3b6⁻) allele of the mouse type IV 3β-HSD encoding gene. The second exon of the gene, which contains the initiation codon (ATG), was flanked with loxP sites. b, Southern blot of ApaI-digested DNA from Hsd3b6^+/+ and Hsd3b6^+/fxneo mice in (a). c, d, Genotyping PCRs. Amplified DNA fragments from Hsd3b6⁺ (0.7 kb), Hsd3b6^fx (1.1 kb), and Hsd3b6⁻ (0.4 kb) alleles are indicated. e, Whole-mount meibomian gland staining of WT and Hsd3b6^−/− female mice. Bars, 1 mm. f, Quantification of the meibomian gland area in (e) (2 mo, n = 10 mice per genotype). Male data are shown in Fig. 2f. g, The number of meibomian gland ducts. n = 8, WT male; n = 10, Hsd3b6^−/− male; n = 10, WT female; n = 10, Hsd3b6^−/− female mice. h, Representative area of gland drop-out of 6-mo Hsd3b6^−/− mouse lower eyelids (n = 2 mice). i, Evaporative dry-eye test for female mice. Photos show representative images of corneal fluorescein staining before and after desiccation (iD). Bars, 0.5 mm. Arrow indicates the area of the epithelial defect. The graphs represent pair-wise comparison of corneal fluorescein staining scores before and after iD of WT and Hsd3b6^−/− female mice (3-4 mo, n = 12, WT; n = 18, Hsd3b6^−/−). Male data are shown in Fig. 2h. ^***P = 0.0006. j, Schimer’s tear test of WT and Hsd3b6^−/− female mice (n = 10, for each genotype). Male data are available in Fig. 2i. Error bars indicate SEM. Statistics in f-g and j: two-way ANOVA, Bonferroni test. Gel images shown in b-d are representative of three independent experiments with similar results. Data in h were reproducibly observed in four independent animals. n.s., not significant. mo, months old.