1. Holocrine Secretion of Sebum Is a Unique DNase2-Dependent Mode of Programmed Cell Death 
Heinz Fischer, Judith Fumicz, Heidemarie Rossiter, Markus Napirei, Maria Buchberger, Erwin Tschachler, Leopold Eckhart 
Journal of Investigative Dermatology 
Volume 137, Issue 3, Pages (March 2017)
DOI: /j.jid
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2. Figure 1
Deletion of Dnase2 suppresses the breakdown of nuclear DNA in differentiating sebocytes. (a) Schematic depiction of terminal differentiation of sebocytes. Mature sebocytes undergo cell death releasing the cellular contents in the form of sebum into the hair channel and onto the hair and skin surface. Intact nuclei are indicated in blue and partially degraded nuclei are shown in red. (b) Quantitative real-time reverse transcriptase–PCR analysis of DNase2 mRNA in keratinocytes and preputial sebaceous glands of wild-type and Dnase2Δep mice. Bars and error bars indicate means and standard deviations, respectively (n = 3). ∗P < (c) Sections of tail skin sebaceous glands of wild-type (n ≥ 20) and Dnase2Δep (n ≥ 20) mice were analyzed by H&E staining and DNA labeling with Hoechst Representative images are shown. Dashed lines denote the border of the glands. The direction of sebocyte differentiation is indicated by arrows. Arrowheads indicate nuclear remnants detected in terminally differentiated sebocytes of Dnase2Δep mice. Nuclear remnants per sebaceous gland (NR/SG) were counted, and mean ± standard deviations are shown below the H&E images. The difference between wild-type and Dnase2Δep mice was significant (∗P < 0.01). Scale bars = 20 μm. a.u., arbitrary unit; H&E, hematoxylin and eosin.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
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3. Figure 2
Abrogation of Atg7-dependent autophagy leads to premature DNA degradation via lysosomal DNase2. Sections of preputial sebaceous glands of wild-type (n ≥ 20), Atg7–/– (n ≥ 20), and Dnase2Δep Atg7Δep (n = 4) mice were analyzed by (a) H&E staining, (b) DNA labeling with Hoechst 33258, and (c) immunofluorescence for LAMP1. Representative images (n = 3 per genotype) are shown. Dashed lines indicate the basal border of sebaceous gland acini. Dotted lines indicate the border between areas with nucleated cells (N) and enucleate cell remnants (E). The ratio of the enucleate area (between the last nucleated cells and the duct) to the total area of the sebaceous gland was determined using Image J software (National Institutes of Health, Bethesda, MD; n>3). Mean ± standard deviations are shown below the H&E images. The differences between all genotypes were significant (P < 0.05). In panel b, the border between sebocyte cellular remnants and the open duct of the sebaceous gland is marked with a full line. In c, arrowheads indicate nuclei aberrantly retained in Dnase2Δep Atg7Δep mice. The direction of sebocyte differentiation is indicated by arrows. Insets in a and b show differentiated sebocytes at higher magnification. Scale bar = 40 μm in a and b, and 20 μm in c. E, enucleate cell remnants; Enucl, enucleate; H&E, hematoxylin and eosin; N, nucleated cells.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
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4. Figure 3
Analysis of nuclear proteins and DNA degradation intermediates in Dnase2Δep mice. Localization of (a) lamin B1 (red), (b) histone H1 (red), and (c) histone H3 (red) by immunofluorescence labeling of sebaceous glands (tail) from Dnase2Δep (n = 5) and control mice (n = 5). Labeling for DNA was performed using Hoechst (blue). Representative images are shown. Nuclear remnants (open arrowheads) strongly positive for histone H3 (arrowheads) were present only in differentiated sebocytes of Dnase2Δep mice. (d) Sections of sebaceous glands from tail skin were analyzed by using TUNEL (red) and DNA (blue) labeling. Nuclear remnants, intensively labeled by TUNEL, were detected in Dnase2Δep (n ≥ 10) mice (arrowheads) but not in sebocytes of wild-type mice (n ≥ 10). The direction of sebocyte differentiation is indicated by arrows. Dashed lines denote the borders of the glands. Scale bars = 20 μm.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
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5. Figure 4
DNA degradation by DNase2 contributes to generation of skin surface uric acid. (a) Extracts from preputial sebaceous glands and sebum from the hair surface were analyzed for acid DNase activity. Two representative samples of wild-type (n = 6) and Dnase2Δep (n = 6) mice are shown. Sebum was further analyzed by quantitative PCR with specific primers for (b) nuclear DNA and (c) mitochondrial DNA. (d) Uric acid was quantified by an enzyme-based assay, and (e) total protein in sebum was determined by the bicinchoninic acid assay. At least five mice were used per genotype. P-values were calculated by the nondirectional two-tailed t test (∗P < 0.05). Dn2, Dnase2; kbp, kilo base pairs; mitoch, mitochondrial; neg. contr., negative control; n.s., not significant; wt, wild-type.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2016 The Authors Terms and Conditions

6. Figure 5
Hypothetical model of cell death during holocrine secretion. Schematic overview of the processes controlling the programmed cell death during sebum production. Events suggested by results of this study are shown.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2016 The Authors Terms and Conditions