SOPten flox/flox (KO) Pten flox/flox (WT) flox allele 6.0 kb. Pten. Actin. ! allele 2.3 kb. Supplementary Figure S1. Yanagi, et al.
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1 s1 A Pten flox/flox () SOPten flox/flox () flox allele 6. kb B Pten flox/flox () SOPten flox/flox () Pten Actin! allele 2.3 kb Supplementary Figure S1. Yanagi, et al.
2 A B BrdU BrdU positive cells ( ) 3 15 * Ks2 TUNEL DAPI TUNEL positive cells ( ) 8 4 Supplementary Figure S2. Yanagi, et al.
3 CD31 -SMA desmin GFAP Supplementary Figure S3. Yanagi, et al.
4 A B Cancer-free survival (%) (n=48) (n=14) Adenocarcinoma n=13 Squamous cell carcinoma n= Time (weeks) p <.1 3 C Supplementary Figure S4. Yanagi, et al. D Number of tumors * Mean tumor diameter (mm) **
5 Supplementary Figure Legends Supplementary Figure S1. Generation of SP-C-rtTA/(tetO) 7 -Cre/Pten flox/flox (SOPten flox/flox ) mice. (A) Genomic Southern blot. DNA (2 µg) extracted from lungs of Pten flox/flox (E1-16) () and SOPten flox/flox (E1-16) () mice was digested with Hind III and hybridized to a previously described probe (24). Lungs from the majority of mice showed deletion of the Pten gene. The floxed (flox allele) and deleted (! allele) Pten alleles are shown. (B) Western blot analysis of Pten protein in lungs of 8- week-old (E1-16) and SOPten flox/flox (E1-16) () mice. Actin, loading control. Supplementary Figure S2. Increased cell proliferation in SOPten flox/flox (E1-16) lung (A) Enhanced proliferation. Left panel: BrdU-positive lung cells (arrows) in lungs of E19.5 (E1-16) and SOPten flox/flox (E1-16) () mice were counted 2 hr after BrdU administration to their dams. Bars, 5 µm. Right panel: data were expressed as the mean number of BrdU-positive lung cells ± SD/1!1 3 cells per mouse in 4 mice/group. *, p<.1, Student s t-test. (B) No effect on apoptosis. Upper left panels: TUNEL positive lung cells (arrows). Lower left panels: control DAPI staining. Bars, 5 µm. Right panel: data were expressed as the mean number of TUNEL-positive lung cells ± SD/1!1 3 cells/mouse in 4 mice/group. Supplementary Figure S3. Increase in number of myofibroblast precursors in the alveolar septa of SOPten flox/flox (E1-16) mice
6 Immunohistochemical analyses of "-SMA, a differentiation marker for smooth muscle cells and myofibroblasts, and desmin, a main component of the intermediate filaments of muscle cells, showed an increased number of "-SMA + desmin - cells in the septa of SOPten flox/flox (E1-16) () lungs. These cells are negative for GFAP, a marker protein for glial cells such as astrocytes. No obvious differences in the intensity or patterns of CD31 expression, a marker protein for vascular endothelial cells, were seen between (E1-16) and (E1-16) mice. Bars, 5 µm. Supplementary Figure S4. Spontaneous lung adenocarcinomas and urethaneinduced lung tumors in SOPten flox/flox (E1-16) mice (A) Gross and histological analyses of spontaneous tumors. Gross appearance of a lung in a 68-week-old (E1-16) mouse and representative lung carcinoma (arrow) in a lung of a 68-week-old SOPten flox/flox (E1-16) () mouse (upper panels). Lower left panel: spontaneous lung adenocarcinoma in a lung of a 72-week-old (E1-16) mouse, representative of tumors observed in 13/14 mice. Lower right panel: squamous cell carcinoma (1/14) in the lung of a 76-week-old (E1-16) mouse. Bars, 5 µm. (B) Kaplan-Meier curves showing cancer-free survival of untreated (E1-16) (n=48) and (E1-16) (n=14) mice. The statistical difference in survival was determined by the log-rank test. (C) Gross and histological analyses of urethaneinduced tumors. (E1-16) (n=26) and (E1-16) mice (n=18) were injected i.p. with urethane. After 2 weeks, tumors were counted and their diameters measured from
7 the lung surface. Upper panels: gross appearance of urethane-induced lung tumors (arrows) in (E1-16) and (E1-16) mice. Lower left panel: histology of a lung adenoma representative of those observed in both (E1-16) and (E1-16) mice. Lower right panel: histology of the one lung adenocarcinoma occurring in a (E1-16) mouse. Bars, 5 µm. (D) Numbers (left panel) and mean size (right panel) of urethane-induced tumors in (E1-16) and (E1-16) mice. The horizontal bars (left panel) represent the mean tumor numbers in the and groups. The right panel shows the mean diameter ± SD of tumors in the and groups. *, p<.5, **, p<.1, Welch s t-test.
8 Supplementary Table S1 Primers for PCR and RT-PCR experiments PCR SP-C-rtTA sense 5'-AAAATCTTGCCAGCTTTCCCC-3' antisense 5'-GACACATATAAGACCCTGGTCA-3' (teto) 7 -Cre sense 5'-TGCCACGACCAAGTGACAGCAATG-3' antisense 5'-AGAGACGGAAATCCATCGCTCG-3' Pten wt and flox allele sense 5'-CTCCCACCAATGAACAAACAGTC-3' antisense 5'-GTGAAAGTGCCCCAACATAAGG-3' K-ras, exon 1 sense 5'-ATGACTGAGTATAAACTTGT-3' antisense 5'-CTCTATCGTAGGGTCGTACT-3' K-ras, exon 2 sense 5'-ACTCCTACAGGAAACAAGT-3' antisense 5'-CTATAATGGTGAATATCTTC-3' K-ras, exon 1, sequence 5'-CTCTATCGTAGGGTCGTACT-3' K-ras, exon 2, sequence 5'-CTATAATGGTGAATATCTTC-3' RT-PCR Spry2 sense 5'-TGTGAGGACTGTGGCAAGTGC-3' antisense 5'-TTTAAGGCAACCCTTGCTGG-3' Shh sense 5'-GAAGATCACAAGAAACTCCGAACG-3' antisense 5'-TGGATTCATAGTAGACCCAGTCGAA-3' FGF7 sense 5'-CAAACGGCTACGAGTGTGAA-3' antisense 5'-TCCGCTGTGTGTCCATTTAG-3' FGF1 sense 5'-GAGAAGAACGGCAAGGTCAG-3' antisense 5'-GGAGGAAGTGAGCAGAGGTG-3' FGFR2 sense 5'-AAATACCAAATCTCCCAACC-3' antisense 5'-GCCGCTTCTCCATCTTCT-3' BMP4 sense 5'-AGGAGGAGGAGGAAGAGCAG-3' antisense 5'-TGTGATGAGGTGTCCAGGAA-3' Wnt7b sense 5'-CCCACATGACAGATGGACAG-3' antisense 5'-GGACCCTGAAGCAGGTATCA-3' Nkx2.1 sense 5'-AACCTGGGCAACATGAGCGAGCTG-3' antisense 5'-ATCTTGACCTGCGTGGGTGTCAGG-3' GATA6 sense 5'-CTTCTAATTCAGATGACTGC-3' antisense 5'-GCACGGAGGATGTGACTTC-3' Nfib sense 5'-CGAACATGGCACGAAAGAGA-3' antisense 5'-TGGACTGGATGGGTCATAAG-3' P3 sense 5'-GTGTCTCCACATGGTGCTTG-3' antisense 5'-GTGTCTCCACATGGTGCTTG-3' CBP sense 5'-CAGAGCGGATCATCCATGACTA-3' antisense 5'-GCTTCTCCATGGTGGCATAC-3' Tcf21 sense 5'-AGCTACATCGCCCACTTGAG-3' antisense 5'-CGGTCACCACTTCTTTCAGG-3' Cutl1 sense 5'-GGACGTAATGTCCGAAGATGC-3' antisense 5'-CTCAGAGGGCGAGGAAATTG-3' Hoxa5 sense 5'-GTGCCAATGTTGTGTGTT-3' antisense 5'-CATACATCACTGAACTGCGC-3'
9 Foxa1 sense 5'-GAAAGGCTAGCCAGCTAGAGG-3' antisense 5'-AGATGCAGCTGAGATTCGTG-3' Foxa2 sense 5'-CCCATTCCAGCGCTTCTC-3' antisense 5'-GTAATGGTGCTCGGGCTTC-3' GR sense 5'-CAAAGCCGTTTCACTGTCC-3' antisense 5'-ACAATTTCACACTGCCACC-3' Adam17 sense 5'-GCGGCGTCTCCTCATCCT-3' antisense 5'-TTATATTCTGCCCCATCTGTGTTG-3' HIF2" sense 5'-GGCGACAATGACAGCTGACAAGGAG-3' antisense 5'-GAGTGAAGTCAAAGATGCTGTGTC-3' enos sense 5'-AAGACAAGGCAGCGGTGGAA-3' antisense 5'-GCAGGGGACAGGAAATAGTT-3' GAPDH sense 5'-TGATGGGTGTGAACCACGAG-3' antisense 5'-GTCATTGAGAGCAATGCCAG-3'
AP VP DLP H&E. p-akt DLP
A B AP VP DLP H&E AP AP VP DLP p-akt wild-type prostate PTEN-null prostate Supplementary Fig. 1. Targeted deletion of PTEN in prostate epithelium resulted in HG-PIN in all three lobes. (A) The anatomy
Probe. Hind III Q,!?R'!! /0!!!!D1"?R'! vector. Homologous recombination
Supple-Zhang Page 1 Wild-type locus Targeting construct Targeted allele Exon Exon3 Exon Probe P1 P P3 FRT FRT loxp loxp neo vector amh I Homologous recombination neo P1 P P3 FLPe recombination Q,!?R'!!
Supplementary Figure 1: Expression of NFAT proteins in Nfat2-deleted B cells (a+b) Protein expression of NFAT2 (a) and NFAT1 (b) in isolated splenic
Supplementary Figure 1: Expression of NFAT proteins in Nfat2-deleted B cells (a+b) Protein expression of NFAT2 (a) and NFAT1 (b) in isolated splenic B cells from WT Nfat2 +/+, TCL1 Nfat2 +/+ and TCL1 Nfat2
Atg5 flox/flox ; CAG-Cre, 19M brain heart lung. spleen stomach colon. Takamura_Fig. S1
Takamura_Fig. S1 brain heart lung spleen stomach colon kidney SM Supplemental Figure 1 Histological findings of tg5 flox/flox ;CG-Cre mouse tissues. H&E staining of the brain, heart, lung, spleen, stomach,
Supplemental Figure S1. RANK expression on human lung cancer cells.
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Supplementary Figure 1. Expression of phospho-sik3 in normal and osteoarthritic articular cartilage in the knee. (a) Semiserial histological sections
Supplementary Figure 1. Expression of phospho-sik3 in normal and osteoarthritic articular cartilage in the knee. (a) Semiserial histological sections of normal cartilage were stained with safranin O-fast
Supplementary Figure 1
Combination index (CI) Supplementary Figure 1 2. 1.5 1. Ishikawa AN3CA Nou-1 Hec-18.5...2.4.6.8 1. Fraction affected (Fa) Supplementary Figure 1. The synergistic effect of PARP inhibitor and PI3K inhibitor
Supplemental Figure 1. Intracranial transduction of a modified ptomo lentiviral vector in the mouse
Supplemental figure legends Supplemental Figure 1. Intracranial transduction of a modified ptomo lentiviral vector in the mouse hippocampus targets GFAP-positive but not NeuN-positive cells. (A) Stereotaxic
Supplemental Table 1. Primers used for RT-PCR analysis of inflammatory cytokines Gene Primer Sequence
Supplemental Table 1. Primers used for RT-PCR analysis of inflammatory cytokines Gene Primer Sequence IL-1α Forward primer 5 -CAAGATGGCCAAAGTTCGTGAC-3' Reverse primer 5 -GTCTCATGAAGTGAGCCATAGC-3 IL-1β
Supplemental Figure 1. (A) The localization of Cre DNA recombinase in the testis of Cyp19a1-Cre mice was detected by immunohistchemical analyses
Supplemental Figure 1. (A) The localization of Cre DNA recombinase in the testis of Cyp19a1-Cre mice was detected by immunohistchemical analyses using an anti-cre antibody; testes at 1 week (left panel),
(A) RT-PCR for components of the Shh/Gli pathway in normal fetus cell (MRC-5) and a
Supplementary figure legends Supplementary Figure 1. Expression of Shh signaling components in a panel of gastric cancer. (A) RT-PCR for components of the Shh/Gli pathway in normal fetus cell (MRC-5) and
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.
Supporting Information Table of Contents
Supporting Information Table of Contents Supporting Information Figure 1 Page 2 Supporting Information Figure 2 Page 4 Supporting Information Figure 3 Page 5 Supporting Information Figure 4 Page 6 Supporting
Supplementary Table 1. List of primers used in this study
Supplementary Table 1. List of primers used in this study Gene Forward primer Reverse primer Rat Met 5 -aggtcgcttcatgcaggt-3 5 -tccggagacacaggatgg-3 Rat Runx1 5 -cctccttgaaccactccact-3 5 -ctggatctgcctggcatc-3
BRaf V600E cooperates with Pten silencing to elicit metastatic melanoma (Nature Genetics Supplementary Information)
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Postn MCM Smad2 fl/fl Postn MCM Smad3 fl/fl Postn MCM Smad2/3 fl/fl. Postn MCM. Tgfbr1/2 fl/fl TAC
A Smad2 fl/fl Smad3 fl/fl Smad2/3 fl/fl Tgfbr1/2 fl/fl 1. mm B Tcf21 MCM Tcf21 MCM Smad3 fl/fl Tcf21 MCM Smad2/3 fl/fl Tcf21 MCM Tgfbr1/2 fl/fl αmhc MCM C 1. mm 1. mm D Smad2 fl/fl Smad3 fl/fl Smad2/3
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
Figure S1. Generation of inducible PTEN deficient mice and the BMMCs (A) B6.129 Pten loxp/loxp mice were mated with B6.
Figure S1. Generation of inducible PTEN deficient mice and the BMMCs (A) B6.129 Pten loxp/loxp mice were mated with B6.129-Gt(ROSA)26Sor tm1(cre/ert2)tyj /J mice. To induce deletion of the Pten locus,
EGFR shrna A: CCGGCGCAAGTGTAAGAAGTGCGAACTCGAGTTCGCACTTCTTACACTTGCG TTTTTG. EGFR shrna B: CCGGAGAATGTGGAATACCTAAGGCTCGAGCCTTAGGTATTCCACATTCTCTT TTTG
Supplementary Methods Sequence of oligonucleotides used for shrna targeting EGFR EGFR shrna were obtained from the Harvard RNAi consortium. The following oligonucleotides (forward primer) were used to
SUPPLEMENTARY INFORMATION
SUPPLEMENTARY INFORMATION Supplementary Figure 1. Generation of a conditional allele of the Kindlin-2 gene. (A) A restriction map of the relevant genomic region of Kindlin-2 (top), the targeting construct
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A. One to three months of age. Anterior Lens (Mean ± SEM) Posterior Lens (Mean ± SEM) Mid Lens (Mean ± SEM) Cornea (Mean ± SEM) Genotype
Supplementary Table 1. Location of lens opacification in Aldh1a1(-/-), Aldh3a1(-/-) single and Aldh1a1(-/-)/Aldh3a1(-/-) double knockout mice (DKO) at different ages. A. One to three months of age Genotype
p = formed with HCI-001 p = Relative # of blood vessels that formed with HCI-002 Control Bevacizumab + 17AAG Bevacizumab 17AAG
A.. Relative # of ECs associated with HCI-001 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 ol b p < 0.001 Relative # of blood vessels that formed with HCI-001 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 l b p = 0.002 Control IHC:
SUPPLEMENTARY INFORMATION
DOI: 10.1038/ncb2607 Figure S1 Elf5 loss promotes EMT in mammary epithelium while Elf5 overexpression inhibits TGFβ induced EMT. (a, c) Different confocal slices through the Z stack image. (b, d) 3D rendering
Supplementary methods:
Supplementary methods: Primers sequences used in real-time PCR analyses: β-actin F: GACCTCTATGCCAACACAGT β-actin [11] R: AGTACTTGCGCTCAGGAGGA MMP13 F: TTCTGGTCTTCTGGCACACGCTTT MMP13 R: CCAAGCTCATGGGCAGCAACAATA
Supplemental Figure 1. Western blot analysis indicated that MIF was detected in the fractions of
Supplemental Figure Legends Supplemental Figure 1. Western blot analysis indicated that was detected in the fractions of plasma membrane and cytosol but not in nuclear fraction isolated from Pkd1 null
(A) PCR primers (arrows) designed to distinguish wild type (P1+P2), targeted (P1+P2) and excised (P1+P3)14-
1 Supplemental Figure Legends Figure S1. Mammary tumors of ErbB2 KI mice with 14-3-3σ ablation have elevated ErbB2 transcript levels and cell proliferation (A) PCR primers (arrows) designed to distinguish
fl/+ KRas;Atg5 fl/+ KRas;Atg5 fl/fl KRas;Atg5 fl/fl KRas;Atg5 Supplementary Figure 1. Gene set enrichment analyses. (a) (b)
KRas;At KRas;At KRas;At KRas;At a b Supplementary Figure 1. Gene set enrichment analyses. (a) GO gene sets (MSigDB v3. c5) enriched in KRas;Atg5 fl/+ as compared to KRas;Atg5 fl/fl tumors using gene set
File Name: Supplementary Information Description: Supplementary Figures and Supplementary Tables. File Name: Peer Review File Description:
File Name: Supplementary Information Description: Supplementary Figures and Supplementary Tables File Name: Peer Review File Description: Primer Name Sequence (5'-3') AT ( C) RT-PCR USP21 F 5'-TTCCCATGGCTCCTTCCACATGAT-3'
Expanded View Figures
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marker. DAPI labels nuclei. Flies were 20 days old. Scale bar is 5 µm. Ctrl is
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E10.5 E18.5 P2 10w 83w NF1 HF1. Sham ISO. Bmi1. H3K9me3. Lung weight (g)
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c Ischemia (30 min) Reperfusion (8 w) Supplementary Figure bp 300 bp Ischemia (30 min) Reperfusion (4 h) Dox 20 mg/kg i.p.
a Marker Ripk3 +/ 5 bp 3 bp b Ischemia (3 min) Reperfusion (4 h) d 2 mg/kg i.p. 1 w 5 w Sacrifice for IF size A subset for echocardiography and morphological analysis c Ischemia (3 min) Reperfusion (8
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Supplemental Figure 1
Supplemental Figure 1 A S100A4: SFLGKRTDEAAFQKLMSNLDSNRDNEVDFQEYCVFLSCIAMMCNEFFEGFPDK Overlap: SF G DE KLM LD N D VDFQEY VFL I M N FF G PD S100A2: SFVGEKVDEEGLKKLMGSLDENSDQQVDFQEYAVFLALITVMCNDFFQGCPDR
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SUPPLEMENTARY FIGURE LEGENDS. atypical adenomatous hyperplasias (AAH); Grade II: adenomas; Grade III: adenocarcinomas;
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a b c periosteum parietal bone bone marrow dura periosteum suture mesenchyme osteogenic front suture mesenchyme 1
coronary suture sagittal suture DOI: 10.1038/ncb3139 a b c e parietal bone suture mesenchyme parietal bone bone marrow ura ura ura f parietal bone ura suture mesenchyme bone g ura osteogenic front suture
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