1. Dual Role of the Anaphase Promoting Complex/Cyclosome in Regulating Stemness and Differentiation in Human Primary Keratinocytes 
Ling Shih Quek, Nicolas Grasset, Joanita Binte Jasmen, Kim S. Robinson, Sophie Bellanger 
Journal of Investigative Dermatology 
Volume 138, Issue 8, Pages (August 2018)
DOI: /j.jid
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2. Figure 1
Cdc20 is exclusively expressed in basal and epibasal layers of the skin epidermis, whereas Cdh1 is amplified in differentiated layers. (a) Staining of the epidermis from skin biopsies (foreskin, 6-year-old male donor) using the indicated antibodies. These results were reproduced using skin biopsies from four different patients (data not shown). Scale bar = 50 μm. (b) Quantification of Cdc20- and Cdh1-positive cells (Cdc20+ and Cdh1+, respectively) in the different layers as indicated. A total of approximately 2,500 cells (from at least four different fields) were counted for each staining. Statistical analyses were performed by applying a two-way analysis of variance. Data represent the means ± standard error of the mean. The proportions of Ki67-negative (Ki67−, gray bars) and of Ki67-positive (Ki67+, striped bars) cells within the Cdc20+ and Cdh1+ populations are shown. Epi, epibasal; H&E, hematoxylin & eosin; ns, not significant.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2018 The Authors Terms and Conditions

3. Figure 2
Expression profiles of Cdc20, Cdh1, E2F1, and ΔNp63α in holoclones, meroclones, and paraclones. (a) Holoclones, meroclones, and paraclones isolated by single-cell cloning (left panel) and later analyzed by western blotting (right panel). Black arrow: Cdh1 expected band (55 kDa), gray arrow: probable Cdh1-modified protein. (b) Quantitative RT-PCR after extraction of mRNA from holoclones, meroclones, and paraclones. Statistical analyses were performed by applying a two-way analysis of variance (n = 9 for holoclones, n = 11 for meroclones, n = 7 for paraclones for K1, K13, Inv, Cdc20, and Cdh1; n = 4 for holoclones, n = 3 for meroclones, n = 3 for paraclones for ΔNp63, E2F1, and Hes1). All data are represented as the means ± standard error of the mean (upper panels), except Cdc20 and Cdh1 data that are shown as a scatter dot plot where each dot represents one clone (lower panel). Inv, involucrin; K1, keratin 1; K13, keratin 13; ns, not significant.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2018 The Authors Terms and Conditions

4. Figure 3
Immunofluorescence experiments showing levels of Cdc20 and Cdh1 in holoclones, meroclones, and paraclones. Immunofluorescence analyses against (a) Cdc20 and Ki67 or (b) Cdh1 and Ki67 performed on holoclones, meroclones, and paraclones grown after single cell isolation. For meroclones, a mix of nondifferentiated and differentiated colonies was present on the dishes, and images that contain the two types of colony in the same field are shown (colonies 1 and 2, respectively).
Journal of Investigative Dermatology  , DOI: ( /j.jid )
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5. Figure 4
Cdc20 silencing induces differentiation, whereas Cdh1 silencing represses differentiation and induces proliferation. (a) Flow cytometry analyses after silencing. One representative experiment is shown (left panel). Statistical analyses (right panel) were performed by applying a two-way analysis of variance (ANOVA). Data represent the means ± standard error of the mean (SEM), n = 3. (b) Clonogenicity tests after silencing. Rhodamine staining is shown (upper panel). Statistical analyses of plating efficiencies (lower-left panel) and of percentages of each colony type (lower-right panel) were performed by applying a one-way ANOVA and paired t-tests, respectively. Data represent the means ± SEM, n = 3. (c) Quantitative real-time PCR analyses after silencing. Statistical analyses were performed by applying one-sample t-tests with a hypothetical value of 1. Data represent the means ± SEM, n = 4. (d) Western-blot analyses after silencing. K1, keratin 1; K10, keratin 10; K13, keratin 13.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2018 The Authors Terms and Conditions

6. Figure 5
Immunofluorescence studies showing the expression of differentiation markers after Cdc20 and Cdh1 silencing. Immunofluorescence analyses performed on cells treated with siRNAs against Cdc20 and Cdh1 as in Figure 4, using antibodies against (a) K10, (b) Flg (two fields are shown for siCdc20), (c) K13/Ki67, and (d) Inv/Ki67. White arrows indicate cells double-positive for proliferation and differentiation markers. Scale bar = 50 μm. Flg, filaggrin; Inv, involucrin; K10, keratin 10; K13, keratin 13.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2018 The Authors Terms and Conditions