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Supplemental Materials and Methods
Animal model
Weekly oral examinations were performed under anesthesia [3-5% isoflurane mixed with
oxygen] to document pathologic changes. Images were recorded using an Olympus SZ40 Stereo
Zoom Microscope at 6.7x magnification and Hitachi KP-D20B CCD color camera. A blinded
observer, scoring images taken during oral examinations, determined disease onset. Survival
rates were estimated from the first date of visible oral tumor formation, using the Kaplan-Meier
method, and differences between the curves were compared using the log-rank test.
Immunohistochemistry
Tissue slides were deparaffinized and rehydrated before performing antigen retrieval in EDTA
buffer at 100C for 30min. Sections were blocked in 10% goat serum in PBS and incubated with
the following primary antibody in blocking buffer at 4C overnight: phospho-p38 MAPK,
phospho-ERK1/2, phospho-SAPK/JNK (Cell Signaling) at 1:1000, isotype controls (Santa Cruz).
After washing in PBS, slides were sequentially incubated with biotinylated secondary antibody
and avidin-biotin complex before developing in 3,3-diaminobenzidine, according the
manufacturers’ protocols. Slides were washed, counterstained with Gill’s hematoxylin,
dehydrated, and mounted. Immunohistochemistry scores were calculated by multiplying the
intensity coefficient and the frequency of positivity coefficient, using the Quick-Score method
(1). The intensity coefficient is scored as 0 (negative), 1 (low), 2 (moderate) or 3 (strong), and
the positivity coefficient is scored based on the percentage of positively staining cells (0 = no
positive staining, 1 = 1-19% positive, 2 = 20-39% positive, 3 = 40-59% positive, 4 = 6079% positive, and 5 = 80-100% positive). The resulting product is categorized from 0 to 5
yielding the IHC staining score as follows: 0 = negative score, 1 = 1-3, 2 = 4-6, 3 = 7-9, 4 = 1012, and 5 = 13-15.
Supplemental Figures
Supplemental Figure 1. Animals underwent weekly oral examinations after carcinogen
withdrawal for the development of oral lesions. Tumor-free survival analysis was performed
using time to first onset of visible tumor growth, as determined by a blinded observer scoring
oral examination images. Dusp1 deficient mice had significantly enhanced tumor onset as
determined by log-rank test. n = 36 per group, p = 0.017.
Supplemental Figure 2. Animals were monitored weekly for weight change as a surrogate
measure for tumor burden. Although Dusp1 deficient mice have been previously characterized as
resistant to diet-induced obesity (2), vehicle-treated wild-type and Dusp1 deficient mice do not
show significant differences in weight change over the course of treatment. n = 15 per group.
Supplemental Figure 3. Macrophage polarization gene expression in tumor tissues by Nanostring
analysis. A, Markers of macrophage M1 polarization revealed no significant differences except
for decreased Il12a expression in Dusp1 deficient tumor tissue. B, Markers of macrophage M2
porlarization revealed significant increases in Arg1, Ym1, and Il4ra expression in Dusp1
deficient tumor tissues with a trend toward increased Cd163 and Cd206 expression. n = 6 per
group, Student’s two-sample t-test, p < 0.05.
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Supplemental Figure 4. Effect of DUSP1 expression on progression-free survival in breast
cancer. Previously generated microarray data was used to stratify patients based on high versus
low DUSP1 expression. Patients with low levels of DUSP1 expression had significantly
decreased time to disease progression. Data generated from model described by Gyorffy et al.
(3).
Supplemental Figure 5. A-C, Immunohistochemistry for phosphorylated forms of the MAPK
p38, JNK, and ERK1/2 were performed on wild-type and Dusp1 deficient tumor samples to
characterize activation of MAPK in epithelial tissues. Staining scores were calculated based on
the product of staining intensity and percentage of staining positivity only within the tumor
epithelium. D, Representative images of IHC staining for phospho-p38 MAPK in oral squamous
cell carcinoma on the tongue are shown. Only phospho-p38 MAPK was elevated in tumor
epithelial tissues compared to vehicle-treated controls for both wild-type and Dusp1 deficient
samples. No difference in staining was seen in tumor epithelium from wild-type and Dusp1
deficient samples. n = 12 (vehicle), 15 (4NQO). Kruskal-Wallis with Dunn’s post-test, p < 0.05.
Supplemental Figure 6. Whole tumor lysates were assessed for phosphorylated and total forms of
p38, JNK, and ERK1/2 MAPKs. Notably, Dusp1 deficient tumor tissues had increased levels of
total MAPKs, rather than phosphorylated forms.
References
1.
Detre S, Saclani Jotti G, Dowsett M. A "quickscore" method for immunohistochemical
semiquantitation: validation for oestrogen receptor in breast carcinomas. J Clin Pathol
1995;48(9):876-8.
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2.
3.
Wu JJ, Roth RJ, Anderson EJ, Hong EG, Lee MK, Choi CS, et al. Mice lacking MAP
kinase phosphatase-1 have enhanced MAP kinase activity and resistance to diet-induced
obesity. Cell Metab 2006;4(1):61-73.
Gyorffy B, Lanczky A, Eklund AC, Denkert C, Budczies J, Li Q, et al. An online
survival analysis tool to rapidly assess the effect of 22,277 genes on breast cancer
prognosis using microarray data of 1,809 patients. Breast Cancer Res Treat
2010;123(3):725-31.
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