Concomitant loss of EAF2/U19 and Pten synergistically promotes prostate carcinogenesis
in the mouse model
Supplementary Materials and Methods
Semi-quantitative analysis of pAkt and p44/42 staining. Analysis was performed on
immunostained murine tissues. A common method of semi-quantitative scoring is referred to as
the histochemical score or simply “H-score.” (1). The H-score provides an overall score (0–300)
based on the sum of ordinal weighted percentiles of cells stained weak, moderate, and strong.
Some institutions, including our own, prefer the H-score because of its wide dynamic range and
use of weighted percentiles. The percentage of prostate epithelial cells that expressed the antigen
was estimated in at least three randomly selected fields at a final magnification of 20X. Staining
intensity was evaluated by two parameters (staining intensity and percentage of cells exhibiting
each level of intensity). Intensity of reaction product was based on a 4-point scale – none,
faint/equivocal, moderate and intense. An H-Score was calculated for each immunostain by cell
type using the following formula: H-Score = 0(% no stain) + 1(% faint/equivocal) + 2(%
moderate) + 3(% intense).
Reference
1.
McCarty KS, Jr., Miller LS, Cox EB, Konrath J, McCarty KS, Sr. Estrogen receptor
analyses. Correlation of biochemical and immunohistochemical methods using monoclonal
antireceptor antibodies. Archives of pathology & laboratory medicine. 1985;109(8):716-21.
Epub 1985/08/01.
Statistical analysis. All statistical analysis was performed using online GraphPad software
(http://www.graphpad.com/). Statistical differences were determined by Chi-square test without
Yates correction in supplementary figure S1, Fisher’s exact test in supplementary figure S6 and
S7, Student’s t test in figure S5 and S10. P < 0.05 was considered significant.
Supplementary figure legends
Supplementary Figure S1. Wet weight of mouse prostates with indicated genotypes at 7 and
19 weeks of age. A, Wet weight of 7-week-old mouse prostates with indicated genotypes. B,
Wet weight of 19-week-old mouse prostates with indicated genotypes. (9 mice per group).
Supplementary Figure S2. Histological changes in mouse prostates at the 7 weeks of age.
Images shown are hematoxylin and eosin (H&E) staining of anterior (AP), dorsal (DP), lateral
(LP) and ventral (VP) prostates of wild-type, EAF2-/-, Pten+/- and EAF2-/-Pten+/- at 7 weeks.
Original magnification 20x. Scale bars represent 100 µm.
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Supplementary Figure S3. Histological changes in mouse prostates at 19 weeks of age.
Images shown are hematoxylin and eosin (H&E) staining of anterior (AP), dorsal (DP), lateral
(LP) and ventral (VP) prostates of wild-type, EAF2-/-, Pten+/- and EAF2-/-Pten+/- at 19 weeks.
Original magnification 20x. Scale bars represent 100 µm.
Supplementary Figure S4. Basal and luminal epithelial cells in EAF2-/-Pten+/- mouse
prostates at 12 months of age. Basal epithelial cells in dorsal prostate (DP) of EAF2-/-Pten+/mouse were immunostainedusing anti-p63 or anti-CK14 antibodies. Luminal epithelial cells in
DP of EAF2-/-Pten+/- mouse were immunostained using anti-AR antibody. Original
magnification 40x. Scale bars represent 50 µm.
Supplementary Figure S5. Semi-quantitative analysis of pAkt and p44/42 staining of
mouse prostates at the age of 12 months. A, Phosphorylated Akt staining in anterior prostate
(AP) and dorsal-lateral prostate (DLP) from intact mice with indicated genotype. B,
Phosphorylated Akt staining in AP and DLP from castrated mice with indicated genotype. C,
Phosphorylated p44/42 staining in AP and DLP from intact mice with indicated genotype. **,
p<0.01, ***, p<0.001
Supplementary Figure S6. Synergistic activation of Akt and p44/42 signaling pathways in
prostate epithelial cells of EAF2-/-Pten+/- mice at 7 weeks. A, Representative phosphorylated
Akt staining in anterior prostate (AP) and dorsal-lateral prostate (DLP) from intact mice with
indicated genotypes at the age of 7 weeks. B, Representative phosphorylated p44/42 staining in
AP and DLP from intact mice with indicated genotypes at the age of 7 weeks. The number in the
images indicates the penetrance of pAkt and p-p44/42 staining. Original magnification 10x and
40x. Scale bars represent 200 µm in 10x and 50 µm in 40x.
Supplementary Figure S7. Synergistic activation of Akt and p44/42 signaling pathways in
prostate epithelial cells of EAF2-/-Pten+/- mice at 19 weeks of age. A, Representative
phosphorylated Akt staining in anterior prostate (AP) and dorsal-lateral prostate (DLP) from
intact mice with indicated genotypes at the age of 19 weeks. B, Representative phosphorylated
p44/42 staining in AP and DLP from intact mice with indicated genotypes at the age of 19 weeks.
The number on the images indicates the penetrance of pAkt and p-p44/42 staining. Original
magnification 10x, inset magnified 40x. Scale bars represent 200 µm in 10x and 50 µm in 40x.
Supplementary Figure S8. Reduction or loss of Pten expression in prostate epithelial cells
of EAF2-/-Pten+/- at the age of 12 months. Immunostaining of Pten was performed in lateral
prostate (LP) of wild-type, EAF2-/-, Pten+/- and EAF2-/-Pten+/- mouse. Arrows indicate
reduced or diminished Pten staining. The number in the images indicates that 4 of 9 EAF2-/Pten+/- mice showed reduction or loss of Pten expression (p=0.0412). Original magnification
40x. Scale bars represent 50 µm.
Supplementary Figure S9. Phospho-JNK expression in prostate epithelial cells of various
genotypes of mice at the age of 12 months. Images shown are representative phosphorylated
JNK staining in anterior prostate (AP) and dorsal-lateral prostate (DLP) from intact mice with
indicated genotypes at the age of 12 months. Original magnification 20x, inset magnified below
40x. Scale bars represent 100 µm in 20x and 50 µm in 40x.
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Supplementary Figure S10. Wnt4 expression in prostate epithelial cells of various
genotypes of mice at the age of 12 months. Images shown are representative Wnt4 staining in
AP and DLP from intact mice with indicated genotypes at the age of 12 months. The number on
the images indicates that 1 of 9 EAF2-/-Pten+/- mice showed enhanced Wnt4 expression (p=0.5).
Original magnification 40x. Scale bars represent 50 µm.
Supplementary Table 1. Gleason score and Ct values of EAF2 and Pten in tumor and
normal adjacent of prostate cancer specimens. Cycle threshold as measured by real-time
qPCR. Fold change was determined by dividing tumor by normal Ct.
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