1. Membrane-Tethered Intracellular Domain of Amphiregulin Promotes Keratinocyte Proliferation 
Stefan W. Stoll, Philip E. Stuart, Sylviane Lambert, Alberto Gandarillas, Laure Rittié, Andrew Johnston, James T. Elder 
Journal of Investigative Dermatology 
Volume 136, Issue 2, Pages (February 2016)
DOI: /j.jid
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2. Figure 1
AREG mRNA and protein expression in AREG subdomain-expressing cell lines. Keratinocytes were grown to 40% confluence and incubated for 48 hours under autocrine conditions with or without Tet. (a and b) AREG mRNA expression in total RNA was analyzed by quantitative real time PCR using two different TaqMan gene expression assays that detect sequences specific to the CTD (a) or the ECD (b) (see Supplementary Figure S1 online). Data are expressed as percentage of RPLP0 transcript levels. Asterisks denote a significant (P < 0.05) reduction in gene expression relative to untreated parental controls, as assessed by two-sample t-test. Bars indicate mean ± SEM, n = 4. AREG protein levels were analyzed by ELISA in cell extracts (c) or in conditioned medium (d). Data are expressed as pg AREG/mg of total protein lysate (n = 3 with two to three biological replicates per experiment). (e) Immunofluorescence staining of AREG in the parental, AREG-CTD, and proAREG expressing cell lines after incubation with or without Tet for 48 hours (scale bar = 25 μm).
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2015 The Authors Terms and Conditions

3. Figure 2
The AREG cytoplasmic domain restores keratinocyte proliferation in AREG knockdown cells. Six-day growth assays were performed with the various AREG cell lines ± Tet in the presence or absence of 100 ng/ml rhAREG. (a) Crystal violet staining of 6-day growth assays with the four keratinocyte cell lines. (b) Quantification of cell number as measured by flow cytometry. Data are expressed as log10 of total cell numbers for the four different treatments indicated underneath the x-axes (n = 4; except EGFP shRNA cell line, n = 3), with three biological replicates per experiment. Data are expressed as mean ± SEM. Asterisks indicate a significant rescue (P < 0.05) for the simple main effect of construct versus the parental cell line. (c) Cell morphology of the various AREG cell lines at the end of 6-day growth assays. Two growth conditions/treatments are shown (AREG, upper panels, and Tet/AREG, lower panels; scale bar = 100 μm).
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2015 The Authors Terms and Conditions

4. Figure 3
Constitutive expression of AREG-CTD does not activate ERK signaling in AREG silenced cells. Parental and AREG-CTD rescue cells were cultured in basal keratinocyte serum-free medium with or without Tet with or without the ErbB tyrosine kinase inhibitor PD (1 μM) or the MEK inhibitor U0126 (10 μM), with and without 100 ng/ml rhAREG. After cell lysis, protein expression was analyzed by Western blotting with antibodies against AREG, phospho-ERK (p-ERK), and total ERK.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
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5. Figure 4
Normalization of cell cycle distribution profiles by proAREG and AREG-CTD. Keratinocyte cell lines were subjected to 6-day growth assays with or without Tet. In all experiments shown, exogenous rhAREG was present at 100 ng/ml; similar effects were observed in the absence of rhAREG (data not shown). (a) Cell cycle distribution of the DAPI-stained parental keratinocyte cell line at the end of the 6-day growth assay. Representative microscope images (on the right) demonstrate differences in cell morphology with the appearance of binucleated cells (arrows) after AREG silencing (scale bar = 100 μm). (b) Mean fluorescence intensities of the major DAPI peaks in the various cell lines with and without Tet (mean ± SEM, n = 4 with two to three replicates per experiment). (c) Quantitation of cell cycle distributions in cell lines cultured in the absence (left panel) or presence of Tet (right panel). Data are expressed as the percentage of cells in G1, S, and G2/M; mean ± SEM (n = 4).
Journal of Investigative Dermatology  , DOI: ( /j.jid )
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6. Figure 5
proAREG and AREG-CTD efficiently restore cell cycle related gene expression after AREG silencing. Keratinocytes were grown to 40% confluence and incubated for 48 hours under autocrine conditions in the presence or absence of Tet. Gene expression was analyzed by quantitative real time PCR using TaqMan gene expression assays. Data are normalized to RPLP0 and expressed for each gene and cell line as percent of no-Tet controls. Asterisks denote that the Tet-induced reduction in mRNA expression for the parental and AREG-ECD constructs is significantly greater than observed for both the proAREG and AREG-CTD constructs (nominal P < 0.05). In contrast, there were no significant reductions in expression for the parental versus the AREG-ECD construct or for proAREG versus the AREG-CTD construct. Data are mean ± SEM (n = 3–4).
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2015 The Authors Terms and Conditions