Supplementary Figure 1. IHC and proliferation analysis of pten-deficient mammary tumors
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1 Wang et al LEGENDS TO SUPPLEMENTARY INFORMATION Supplementary Figure 1. IHC and proliferation analysis of pten-deficient mammary tumors A. Induced expression of estrogen receptor α (ERα) in AME vs PDA tumors. Student s t test, p=.5, n 3. B. Induced expression of Ki67 in PDAs vs AMEs. Student s t test, p=.31, n 3. C. Enlarged immunohistochemistry images of AMEs and PDAs shown in Figure 1I. N=6. D. In vitro growth curves of independent AME tumor cell lines incubated with indicated concentrations of β-estradiol. Student s t test, *p<.5, **p<.1; ***p<.1, n=2. Supplementary Figure 2. Proliferation and apoptosis analyses of AME and PDA cells following orthotopic transplantation into NOD/SCID mice Shown are representative IHC images of phospho-h3, Ki67 and activated caspase 3 of mammary glands injected with AME or PDA cells at indicated time points. n 3. Supplementary Figure 3. GSEA reveals distinct pathways in PDAs versus AMEs Complete GSEA of AMEs (blue) versus PDAs (red) using canonical pathways (c2.all, v4., Broad Institute) visualized using Cytoscape Enrichment Map. Green lines connect overlapping pathways. Size of the circles corresponds with levels of enrichment (normalized enrichment score) whereas thickness of lines corresponds with degree of overlap. p<.1, FDR<.1. PDA n=8, AME n=13. Supplementary Figure 4. g:profiler analysis of AMEs versus PDAs. p<.5. PDA n=8, AME n=13. 61
2 Wang et al Supplementary Figure 5. acgh analysis showing distinct genetic alterations in PDAs versus AMEs Shown is a list of genes with significant copy number changes in PDAs versus AMEs including chromosomal location and frequency of alterations. PDA n=5, AME n=5. Supplementary Figure 6. Schematic representation of Cul9 (a) and Cbl (b), and relative location (asterisks) of mutations identified in PDAs. PDA n=7, AME n=3. Supplementary Figure 7. Kaplan-Meier survival analysis for patients with mir-145-low/pten-low expression showing a trend toward significantly worse DFS compared to patients with mir-145- high/pten-low expression. Supplementary Figure 8. mir-143/145 knockdown cooperates with pten loss by activating the Ras pathway A. In vitro growth curves of independent AME tumor cell lines transduced with mir-143/145 decoy. n 5. B. Epithelial marker analysis of AME and PDA tumors for ERα, K18 and K14. n 3. C. Representative Western Blot analysis of PTEN knockdown by lentiviral shrna. n 3. D. In vitro growth curves of HC11 cells transduced with mir-143/145 decoy, pten shrna or both. n 3. E. Western blot quantification showing elevated phospho-erk levels in HC11 cells treated with both mir-143/145 decoy and pten shrna compared to cells transduced with only mir- 143/145 decoy, or only pten shrna. n 8. 62
3 Wang et al F. Quantitative RT-PCR analysis of indicated predicted mir-143/145 targets following mir- 143/145 overexpression. n=3. Supplementary Figure 9. Distinct levels of activation of 18 signaling pathways in AMEs vs PDAs * denotes significant differences. Red induction in PDAs; green induction in AMEs. PDA n=8, AME n=13. 63
4 Vimentin ERα K14/K18/DAPI K5 Ki67 Her2 % ER positive cells %Ki67 positive cells Figure S1 A p=.5 Data 5 B p= PDA AME 2 1 PDA AME C AME PDA AME PDA AME
5 Cell growth Cell growth D.8 * ** * * **.3 *** estradiol (nm) estradiol (nm)
6 Day 27 Day 6 Day 3 Day 18 Day 6 Day 3 Figure S2 Phospho-H3 Ki67 Activated caspase 3 AME PDA
7 Figure S3
8 Figure S4
9 Figure S5 Region Event Freq. in Freq. in PDA (%) AME (%) Gene Symbols chr4:14,989,635-14,992,922 CN Loss 8 Gm694 chr6:125,678,52-125,697,798 CN Gain 8 Ano2 chr6:13,334,179-13,413,964 CN Gain 8 Klra1 chr6:137,128, ,132,525 CN Gain 8 chr6:137,792, ,87,44 CN Gain 8 chr12:79,949,645-79,962,242 CN Gain 8 Atp6v1d chr12:114,659, ,668,491 CN Loss 8 Igh-A (1g2), Gm16844, Igh, Ighg, V821, abparts chr12:114,668, ,676,477 CN Loss 1 abparts chr12:114,715, ,722,266 CN Gain 8 abparts chr12:117,668,3-117,679,572 CN Gain 2 1 Ncapg2 chr13:36,129,995-36,14,576 CN Loss 8 Lyrm4 chr14:51,574,375-51,586,687 CN Gain 8 Pnp2 chr14:52,494,38-52,52,775 CN Gain 8 chr14:52,575,12-52,593,14 CN Gain 8 chr19:17,165,722-17,173,735 CN Loss 8 Prune2
10 Figure S6 A Cul9 E2467 E2467 E2467 E2467 N Cul17 APC1 Cullin IBR * C B N CBL G848D S873T A895T G848D S873T A895T A895T S873T * * * TKB domain RING Proline-rich UBA/LZ C
11 Figure S7 Disease free survival A Months Mir145-high:PTEN-high n = 19 Mir145-low:PTEN-high n = 42 Mir145-high:PTEN-low n = 15 Mir145-low:PTEN-low n = 13 HR: 1.8 P =.248 HR: 1.94 P =.685 HR: 2.39 P =.55
12 Relative Expression AME+ Empty Vector Relative cell number AME+ mir Decoy Relative cell number Relative cell number A B Figure S p< p<.1 6 p< p<.1 p<.1 2 Day 1 Day 2 Day 3 Day 4 Day 1 Day 2 Day 3 Day 4 ERα K K14 Day 1 Day 2 Day 3 Day 4 D p<.5 15 p<.1 8 p<.1 p<.1 Day 1 Day 2 Day 3 Day C Day 1 Day 2 Day 3 Day 4 Pten shrna Pten - + Empty vector + - Tubulin PTEN shrna+mirna Decoy mirna Decoy PTEN shrna p< Empty Vector p<.43 p<.42 2 E Double infection vs. Pten shrna Double infection vs. mir Decoy 1 5 Day 1 Day 2 Day 3 Day Efna1b Fli1 Gng7 Rasal1 F 25 2 Empty Vector mir overexpression
13 Figure S9 Pathway AMEs PDAs p value AKT β-catenin E2F EGFR ER* E-5 Her IFNα* IFN gamma Myc p p PI3K PR Ras* Src STAT TGFβ* TNFα*
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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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Tbk1-TKO! DN cells (%)! 15! 10!
a! T Cells! TKO! B Cells! TKO! b! CD4! 8.9 85.2 3.4 2.88 CD8! Tbk1-TKO! 1.1 84.8 2.51 2.54 c! DN cells (%)! 4 3 2 1 DP cells (%)! 9 8 7 6 CD4 + SP cells (%)! 5 4 3 2 1 5 TKO! TKO! TKO! TKO! 15 1 5 CD8
Supplemental Information
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Supplementary Figure 1
A B D Relative TAp73 mrna p73 Supplementary Figure 1 25 2 15 1 5 p63 _-tub. MDA-468 HCC1143 HCC38 SUM149 MDA-468 HCC1143 HCC38 SUM149 HCC-1937 MDA-MB-468 ΔNp63_ TAp73_ TAp73β E C Relative ΔNp63 mrna TAp73
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doi:10.1038/nature12652 Supplementary Figure 1. PRDM16 interacts with endogenous EHMT1 in brown adipocytes. Immunoprecipitation of PRDM16 complex by flag antibody (M2) followed by Western blot analysis
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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
File Name: Supplementary Information Description: Supplementary Figures and Supplementary Table. File Name: Peer Review File Description:
File Name: Supplementary Information Description: Supplementary Figures and Supplementary Table File Name: Peer Review File Description: Supplementary Table 1 Primers and taqman probes used were the following:
Supplemental Figure 1. Signature gene expression in in vitro differentiated Th0, Th1, Th2, Th17 and Treg cells. (A) Naïve CD4 + T cells were cultured
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Supplementary Figure 1
Supplementary Figure 1 14 12 SEM4C PLXN2 8 SEM4C C 3 Cancer Cell Non Cancer Cell Expression 1 8 6 6 4 log2 ratio Expression 2 1 4 2 2 p value.1 D Supplementary Figure 1. Expression of Sema4C and Plexin2
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doi:10.1038/nature12215 Supplementary Figure 1. The effects of full and dissociated GR agonists in supporting BFU-E self-renewal divisions. BFU-Es were cultured in self-renewal medium with indicated GR
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