Figure 2.

Deletion of embigin disrupts sebaceous gland homeostasis

(A) Hematoxylin and eosin staining of the tail skin of adult WT and Emb^−/− mice.

(B) Adult tail wholemounts from WT and Emb^−/− as well as from Emb^flox/flox and K14CreEmb^flox/flox mice labeled with antibodies against ITGA6 (green) and DAPI (blue).

(C) Quantification of area covered by SGs per HF. The average of ≥9 HFs per mouse quantified from wholemounts is shown (Cre-, n = 4 mice; K14CreEmb^flox/flox, n = 4 mice; WT, n = 4 mice; Emb^−/−, n = 4 mice).

(D) Quantification of size of suprabasal sebocytes (WT, n = 72, Emb^−/−, n = 72) from Herovici stained sections. Cells were pooled from 3 WT and 3 Emb^−/− mice. Red circles delineate examples of individual suprabasal cells.

(E) Quantification of suprabasal sebocyte number per sebaceous gland from cryosections. Average ± SD of mice is shown (2–3-month WT, n = 5; 2–3-month Emb^−/−, n = 4; 7-month WT, n = 3; 7-month Emb^−/−, n = 3). At least 7 SGs per mouse were quantified.

(F) Tail wholemounts from WT and Emb^−/− mice labeled with antibodies against Ki67 (red) and ITGA6 (green) with DAPI nuclear counterstain (blue).

(G) Quantification of Ki67+ cells in WT and Emb^−/− SGs (per) SG or normalized to SG size (WT, n = 37; Emb^−/−, n = 28). SGs were pooled from 3 WT and 3 Emb^−/− mice.

(H) Cryosections of Cre- and K14CreEmb^flox/flox mouse tail SG on the same day (0d) or 11 days after (11d) EdU injection stained with antibodies to EdU (green) and basement membrane marker laminin (red) with DAPI nuclear counterstain (blue).

(I) Quantification of the percentage of suprabasal EdU+ cells per SG (0-day Cre-, n = 30; 0-day K14 K14CreEmb^flox/flox, n = 32; 11-day Cre-, n = 48; 11-day K14CreEmb^flox/flox, n = 45). SGs were pooled from 3 mice per group.

(J) Cryosections of neonatal (P1–P2) WT and Emb^−/− tail HFs stained for stearoyl-CoA desaturase-1 (Scd1), which is involved in lipid synthesis (green), and basement membrane marker laminin (red) with DAPI nuclear counterstain (blue).

(K) Quantification of SCD1+ cell number per stage V–VI HF (WT, n = 48; Emb^−/−, n = 35). HFs were pooled from 4 WT and 3 Emb^−/− mice. In stage V–VI HF, sebocytes can be detected and start forming a sebaceous gland but the SG is not yet localized on the HF posterior wall (Paus et al., 1999).

(L) Cryosections of WT and Emb^−/− tail skin stained with a lipid marker LipidTOX (green) and ITGA6 (red) with DAPI nuclear counterstain (blue).

(M) Quantification of SG lipids (LipidTOX pixels) in WT and Emb^−/− SGs (WT, n = 30; Emb^−/−, n = 18). SGs were pooled from 6 WT and 4 Emb^−/− mice.

(N) Quantification of total lipids on mouse skin surface by hexane extraction and TLC (WT, n = 3 mice; Emb^−/−, n = 3 mice).

Two-tailed t test (C, D, G, I, K, M, and N) or one-tailed t test (E) for independent means was used to determine statistical significance. ^∗∗∗p < 0.0005, ^∗∗p < 0.005, ^∗p < 0.05. The length of scale bars is 50 μm.