1. Volume 2, Issue 2, Pages 180-188 (February 2014)
Conserved Two-Step Regulatory Mechanism of Human Epithelial Differentiation 
Jayant K. Rane, Alastair P. Droop, Davide Pellacani, Euan S. Polson, Matthew S. Simms, Anne T. Collins, Leo S.D. Caves, Norman J. Maitland 
Stem Cell Reports 
Volume 2, Issue 2, Pages (February 2014)
DOI: /j.stemcr
Copyright © 2014 The Authors Terms and Conditions

2. Stem Cell Reports 2014 2, 180-188DOI: (10.1016/j.stemcr.2014.01.001)
Copyright © 2014 The Authors Terms and Conditions

3. Figure 1 Identification of Coregulated Genes
(A) Selection of differentially expressed probes between SC and CB (p < 0.01; log2 fold change ≥ 0.75) are represented as black dots in the Birnie et al. (2008) microarray data.
(B) Differentially expressed probes were clustered in distinct A–D groups on coexpression analysis. Principal GO terms enriched for groups B and C are shown below the group name. Group A was not enriched for any GO terminology, whereas group C was enriched for genes involved in (lens) development.
(C) Normalized expression of group A–D probes. Probes overexpressed in committed basal cells (CB) are above the horizontal line at 0 (groups A and C) and probes overexpressed in stem-like cells (SC) (groups B and D) are below the line. Boxplot shows minimum, 25%, median, 75%, and maximum.
Stem Cell Reports 2014 2, DOI: ( /j.stemcr )
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4. Figure 2
Genes in Groups A–D Are Coexpressed in the Majority of Human Cell Types
(A) Overview of the composition of compendium data set.
(B) Schematic representation of the methodology for the compendium data set.
(C) Correlation of coexpression of genes in groups A–D compared to all genes in the compendium data set. All the groups are significantly different where the p value is computationally indistinguishable from zero.
(D) Expression of candidate group C genes in the Birnie et al. (2008) microarray data. BS, benign stem-like cells; MS, malignant stem-like cells; BC, benign committed basal cells; MC, malignant committed basal cells.
(E) Western blot analysis of LCN2, CEACAM6, and S100P expression. Representative image, n = 6 each for benign prostatic hyperplasia (BPH) and treatment-naïve prostate cancer (PCa) (biological replicates).
(F) Rainbow contour graphs for the correlation of coexpression for TMPRSS2, CEACAM6, LCN2, and S100P with each other in compendium data set. Values at the top of each square indicate correlation coefficient. All values are significant with p < 0.05.
All boxplots show minimum, 25%, median, 75%, and maximum. See also Figure S1 and Table S1.
Stem Cell Reports 2014 2, DOI: ( /j.stemcr )
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5. Figure 3
Retinoic Acid Can Regulate Group C while Inducing Early Prostate Stem Cell Differentiation
(A) Schematic representation for selection process of retinoic acid (RA) as a group C regulator.
(B) Expression of RA receptors in primary prostate epithelial cultures (PPECs) by immunofluorescence (representative image); n = 3 each for BPH and PCa (biological replicates). Scale bar: 62 μm.
(C–F) qRT-PCR analysis of CEACAM6, LCN2, S100P, and TMPRSS2 expression after 100 nM at-RA treatment of PPECs; n = 5 for BPH (3) and PCa (2) (biological replicates).
(G) qRT-PCR analysis of CEACAM6, LCN2, S100P, and TMPRSS2 expression after 100 nM at-RA treatment of SCs; mean values shown for BPH (five) and PCa (four) (biological replicates).
(H) Changes in the expression of group C genes after RA treatment in four published microarray experiments.
(I) Changes in colony-forming efficiency of PPECs after 72 hr 100 nM at-RA treatment; n = 4 for BPH and PCa.
(J and K) Fluorescence-activated cell sorting analysis of CD49f expression after 72 hr treatment of PPECs with 100 nM at-RA; n = 4 each for BPH and PCa (biological replicates). In (J), the blue line indicates DMSO control, whereas the red line indicates RA-treated samples. Dotted lines represent unlabeled controls.
(L) Immunofluorescence (IF) analysis of KRT5 and KRT18 after 72 hr treatment of PPECs with 100 nM at-RA. Scale bar, 100 μm.
(M and N) Quantification of changes in KRT5 (M) and KRT18 (N) protein expression by IF after 72 hr treatment of PPECs with 100 nM at-RA using velocity software.
(O) qRT-PCR expression analysis of markers of prostate epithelial differentiation after 72 hr treatment of PPECs with 100 nM at-RA; n = 4 each for BPH and PCa (biological replicates).
Error bars represent mean ± SD. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < Box plots in (H), (M), and (N) show minimum, 25%, median, 75%, and maximum. See also Figure S2.
Stem Cell Reports 2014 2, DOI: ( /j.stemcr )
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6. Figure 4
Androgens Regulate Group C Gene Expression during Terminal Differentiation
(A) Expression of group A–D genes in prostate luminal cells relative to basal cells.
(B) Changes in expression of group C genes after androgen stimulation in three published microarrays.
(C) qRT-PCR analysis of TMPRSS2 expression after 48 hr treatment with at-RA (RA) and R1881 (n = 6 for PPECs [3 BPH and 3 PCa, biological replicates] and n = 3 for cell lines [experimental replicates]).
(D) qRT-PCR for the expression of LCN2, CEACAM6, S100P, TMPRSS2, and PSA in LNCaP cells after 1 nM bicalutamide treatment for 48 hr. The data are plotted considering the untreated gene expression value as 1 (n = 3, experimental replicates).
Error bars represent mean ± SD. Box plots show minimum, 25%, median, 75%, and maximum. See also Figures S3 and S4.
Stem Cell Reports 2014 2, DOI: ( /j.stemcr )
Copyright © 2014 The Authors Terms and Conditions