BRaf V600E cooperates with Pten silencing to elicit metastatic melanoma (Nature Genetics Supplementary Information)
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1 BRaf V600E cooperates with Pten silencing to elicit metastatic melanoma (Nature Genetics Supplementary Information) David Dankort, David P. Curley, Robert A. Cartlidge, Betsy Nelson, Anthony N. Karnezis, William E. Damsky, Jr., Mingjian J. You, Ronald A. DePinho, Martin McMahon & Marcus Bosenberg Supplementary Table 1. Lethality of Tyr::Cre; BRaf CA mice BRaf CA/+ and BRaf CA/CA mice were crossed to Tyr::Cre transgenic mice (Tec1). The resulting progeny were analyzed for the presence of the BRaf + or BRaf CA alleles and the Tyr::Cre transgene by PCR. No Tyr::Cre; BRaf CA mice were obtained in these crosses. Supplementary Table 2. PCR primers used for genotyping Pten lox5 mice Supplementary Figure 1. Induction of benign melanocytic lesions in BRaf CA/+ mice requires the Tyr::CreER transgene (a) The ears of neonatal BRaf CA/+ (left) and Tyr::CreER; BRaf CA/+ (right) mice were treated with 4-HT and then monitored for 7 weeks for the development of benign melanocytic lesions. The feet of the 4- HT treated BRaf CA/+ (b) and Tyr::CreER; BRaf CA/+ (c) mice developed benign melanocytic lesions, most likely due to grooming behavior. Supplementary Figure 2. Melanomas arising in Tyr::CreER; BRaf CA/+ ; Pten lox4-5/+ mice Adult Tyr::CreER mice with the indicated genotypes were treated with 4-HT on the right ear, right flank and distal tail and monitored for the development of melanoma. Kaplan-Meier survival analysis of 4-HT treated: 1. Tyr::CreER; BRaf CA/+ ; Pten +/+ mice (n=22); 2. Tyr::CreER; BRaf +/+ ; Pten lox4-5/lox4-5 mice (n=5); 3. Tyr::CreER; BRaf CA/+ ; Pten lox4-5/lox4-5 mice (n=22) and; 4. Tyr::CreER; BRaf CA/+ ; Pten lox4-5/+ (n=5). Log rank tests of survival plots of the data indicated a statistically significant difference between the following survival curves: Tyr::CreER; BRaf CA/+ ; Pten n lox4-5/lox4-5/+ versus Tyr::CreER; BRaf CA/+ ; Pten lox4-5/lox4-5 (p<0.0001) and Tyr::CreER; BRaf +/+ ; Pten lox4-5/+ versus Tyr::CreER; BRaf CA/+ ; Pten +/+ (p<0.0001). Melanomas that arose were solitary in nature and were observed only where 4-HT was administered on the ear (b-d), the tail (e & f) and the right flank (g-i). Supplementary Figure 3. Confluent melanocytic proliferation and localized malignant melanoma induction in Tyr::CreER; BRaf CA/+ ; Pten lox5/lox5 mice Adult Tyr::CreER; BRaf CA/+ ; Pten lox5/lox5 mice were treated with 5µl of 5nM 4-HT in a solution of 100% Nature Genetics: doi: /ng.356
2 ethanol either on skin of the back (a), the right ear (b) or the distal tail (c). Mice were monitored for weeks prior to photography of the ear and flank melanoma and 6 months for photography of the tail melanoma. The fur of the mouse in (a) was removed using Veet prior to treatment with 4-HT and again prior to photography. Supplementary Figure 4. Cre-mediated silencing of Pten expression in tumors derived from Tyr::CreER; BRaf CA/+ ; Pten lox5/lox5 mice Malignant melanoma was initiated in Tyr::CreER; BRaf CA/+ ; Pten lox5/lox5 mice by treatment with 4- HT. Melanoma-bearing mice were euthanized and tumor lysates were prepared for analysis of Pten expression by Western blotting. Extracts of NIH 3T3 cells were used as a positive control for Pten expression and Actin was used as a loading control for all lanes. Supplementary Figure 5. Cre-mediated recombination of BRaf CA and Pten lox5 in 2697T cells DNA was isolated from 2697T cells and the toe of the mouse from which the cell line was derived. PCR analysis was used to confirm the rearrangement of the Pten lox5 (homozygous) and BRaf CA (heterozygous) alleles. Supplementary Figure 6. Induction of pyknotic nuclei in Tyr::CreER; BRaf V600E ; Pten null melanoma by treatment with PD325901, Rapamycin or the combination of both agents. Tyr::CreER; BRaf CA ; Pten lox5/lox5 mice with readily measurable melanoma lesions were randomly divided into 4 groups and administered either solvent control (Vehicle), PD (PD), Rapamycin (Rapa) or the combination of both PD and Rapamycin (PD+Rapa) for 3 weeks as indicated in the legend to Figure 6. Five separate fields of H&E stained paraffin tumor sections were quantified for the presence of pyknotic nuclei per square millimeter by visual inspection. Results are presented as a bar graph indicating the average number of pyknotic nuclei ± SEM with p values where significance was defined by unpaired t tests (Prism Graph). Nature Genetics: doi: /ng.356
3 Supplementary Table 1. Lethality of Tyr::Cre; BRaf CA mice BRaf CA/+ and BRaf CA/CA mice were crossed to Tyr::Cre transgenic mice (Tec1). The resulting progeny were analyzed for the presence of the BRaf + or BRaf CA alleles and the Tyr::Cre transgene by PCR. No Tyr::Cre; BRaf CA mice were obtained in these crosses. Offspring Mouse Crosses BRaf CA/+ ; -/- BRaf CA/+ ; Tec1/- BRaf +/+ ; -/- BRaf +/+ ; Tec1/- p-value BRaf CA/+ ; -/- X +/+ ; Tec1/- 31/25 0/25 36/25 34/25 1.8E-7 BRaf CA/CA ; -/- X +/+ ; Tec1/- 21/10 0/ E-6 BRaf CA/+ ; -/- X +/+ ; Tec1/Tec1-0/11-22/11 2.7E-6 The indicated crosses were carried out. The potential genotypes of the offspring are indicated with shaded regions representing genotypes not possible in these matings. The observed /expected number of offspring of the indicated genotypes are depicted as is the Chi-square derived p-value. Supplementary Table 2. PCR primers for genotyping for the presence of the Pten lox5 allele PCR primers for genotyping for the presence of the Pten lox5 allele Pten lox5 primer 1 5'-CTTCGGAGCATGTCTGGCAATGC-3' Pten lox5 primer 2 5'-CTTCGGAGCATGTCTGGCAATGC-3' Pten lox5 primer 2 5'-CTTCGGAGCATGTCTGGCAATGC-3' Nature Genetics: doi: /ng.356
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Supplementary Figure 1 Overview of the transplant procedure and supplementary data to Figure 1. a. Under isofluorane anesthesia, the lumen of the colon is washed by a gentle PBS enema. b. Using a p200
Supplementary. properties of. network types. randomly sampled. subsets (75%
Supplementary Information Gene co-expression network analysis reveals common system-level prognostic genes across cancer types properties of Supplementary Figure 1 The robustness and overlap of prognostic
Supplementary Materials
Supplementary Materials Supplementary figure 1. Taxonomic representation summarized at genus level. Fecal microbiota from a separate set of Jackson and Harlan mice prior to irradiation. A taxon was included
Nature Immunology: doi: /ni Supplementary Figure 1
Supplementary Figure 1 NLRP12 is downregulated in biopsy samples from patients with active ulcerative colitis (UC). (a-g) NLRP12 expression in 7 UC mrna profiling studies deposited in NCBI GEO database.
Reprogramming Tumor Associated Dendritic Cells for Immunotherapy
Reprogramming Tumor Associated Dendritic Cells for Immunotherapy Edgar Engleman, M.D. Professor of Pathology and Medicine Stanford University Disclosures: Founder of Dendreon, a biotechnology company that
Supplementary Figures
Supplementary Figures Supplementary Fig. 1. Galectin-3 is present within tumors. (A) mrna expression levels of Lgals3 (galectin-3) and Lgals8 (galectin-8) in the four classes of cell lines as determined
Supplemental Material
Supplemental Material Supplementary Fig. 1. EETs stimulate primary tumor growth. a) Schematic presentation of genetic and pharmacological tools used to manipulate endogenous EET levels. b) Endothelial
Supplementary Figure 1. Baf60c and baf180 are induced during cardiac regeneration in zebrafish. RNA in situ hybridization was performed on paraffin
Supplementary Figure 1. Baf60c and baf180 are induced during cardiac regeneration in zebrafish. RNA in situ hybridization was performed on paraffin sections from sham-operated adult hearts (a and i) and
PREPARED FOR: U.S. Army Medical Research and Materiel Command Fort Detrick, Maryland
AD Award Number: W81XWH-4-1-82 TITLE: Effect of Saw Palmetto on the Development and Progression of Prostate Carcinoma in TRAMP Mice PRINCIPAL INVESTIGATOR: Charles E. Roselli CONTRACTING ORGANIZATION:
SUPPLEMENTARY FIGURES
SUPPLEMENTARY FIGURES Figure S1. Effect of a HFD on the Acox gene expression in the livers of WT and IL-6 -/- mice. Expression of Acox in the livers of WT and IL-6 -/- mice fed STD or HFD determined through
Nature Structural & Molecular Biology: doi: /nsmb Supplementary Figure 1. Generation and validation of mtef4-knockout mice.
Supplementary Figure 1 Generation and validation of mtef4-knockout mice. (a) Alignment of EF4 (E. coli) with mouse, yeast and human EF4. (b) Domain structures of mouse mtef4 compared to those of EF4 (E.
Plasma exposure levels from individual mice 4 hours post IP administration at the
Supplemental Figure Legends Figure S1. Plasma exposure levels of MKC-3946 in mice. Plasma exposure levels from individual mice 4 hours post IP administration at the indicated dose mg/kg. Data represent
Supplementary Figure 1. DNA methylation of the adiponectin promoter R1, Pparg2, and Tnfa promoter in adipocytes is not affected by obesity.
Supplementary Figure 1. DNA methylation of the adiponectin promoter R1, Pparg2, and Tnfa promoter in adipocytes is not affected by obesity. (a) Relative amounts of adiponectin, Ppar 2, C/ebp, and Tnf mrna
Loss of RhoA promotes skin tumor formation. Supplementary Figure 1. Loss of RhoA does not impair F-actin organization.
Supplementary Figure Legends Supplementary Figure 1. Loss of RhoA does not impair F-actin organization. a. Representative IF images of F-actin staining of big and small control (left) and RhoA ko tumors
Supplementary Figure 1: Hsp60 / IEC mice are embryonically lethal (A) Light microscopic pictures show mouse embryos at developmental stage E12.
Supplementary Figure 1: Hsp60 / IEC mice are embryonically lethal (A) Light microscopic pictures show mouse embryos at developmental stage E12.5 and E13.5 prepared from uteri of dams and subsequently genotyped.
a b G75 G60 Sw-2 Sw-1 Supplementary Figure 1. Structure predictions by I-TASSER Server.
a b G75 2 2 G60 Sw-2 Sw-1 Supplementary Figure 1. Structure predictions by I-TASSER Server. a. Overlay of top 10 models generated by I-TASSER illustrates the potential effect of 7 amino acid insertion
SUPPLEMENTARY FIGURES
SUPPLEMENTARY FIGURES 1 Supplementary Figure 1, Adult hippocampal QNPs and TAPs uniformly express REST a-b) Confocal images of adult hippocampal mouse sections showing GFAP (green), Sox2 (red), and REST
GFP-LC3 +/+ CLU -/- kda CLU GFP. Actin. GFP-LC3 +/+ CLU -/- kda CLU GFP. Actin
Supplementary Fig. 1 a CQ treatment ScrB OGX11 MG132 I II AZD5363 I II b GFP / / GFP / / GFP / / GFP / / GFP GFP Actin Actin ctrl CQ GFP / / GFP / / GFP / / GFP / / GFP GFP Actin Actin rapamycin rapamycincq
Supplementary Information
Supplementary Information Akt regulates hepatic metabolism by suppressing a Foxo1 dependent global inhibition of adaptation to nutrient intake Mingjian Lu 1, Min Wan 1, Karla F. Leavens 1, Qingwei Chu
marker. DAPI labels nuclei. Flies were 20 days old. Scale bar is 5 µm. Ctrl is
Supplementary Figure 1. (a) Nos is detected in glial cells in both control and GFAP R79H transgenic flies (arrows), but not in deletion mutant Nos Δ15 animals. Repo is a glial cell marker. DAPI labels