Supplementary Figure 1 Cell line TRIB2 status. Supplementary Figure 2 TRIB2 status has no impact on the cell cycle after PI3K inhibition. a. b.
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1 Supplementary Figure 1 Cell line TRIB2 status. TRIB2 protein expression to determine endogenous expression and to determine the effectiveness of each of our TRIB2 knockdown constructs. Supplementary Figure 2 TRIB2 status has no impact on the cell cycle after PI3K inhibition. a. Matched isogenic TRIB2 cell line cell cycle analysis via FACS following exposure to BEZ235 (n = 6). Data represent the mean ± standard deviation (s.d.). b. Matched isogenic TRIB2 cell line cell cycle analysis via FACS following exposure to BAY236 (n=6). Data represent the mean ± standard deviation (s.d.). c. Matched isogenic TRIB2 cell line cell cycle analysis via FACS following exposure to BAY439 (n=6). Data represent the mean ± standard deviation (s.d.).
2 Supplementary Figure 3 TRIB2-dependent resistance to BEZ235 is ablated in the presence of a phosphomimetic AKT473D or FOX3a-AAA. Matched isogenic TRIB2 cell line FACS analysis following 72 hour exposure to BEZ235 (n=6), following transient transfection of each indicated plasmid construct. P values are indicated for each comparison by two-way analysis of variance (ANOVA) and data represent the mean ± standard deviation (s.d.).
3 Supplementary Figure 4 PDK1, p70s6k and pser792 Raptor protein levels are not significantly different in cell lines with elevated TRIB2 protein expression. Representative immunoblot profiles for matched isogenic U2OS osteoarcoma cell lines for total PDK1, pser241-pdk1, pser792-raptor, total p70s6k and pthr389-p70s6k. TRIB2 expression did not correlate with significantly different posttranslational modification(s) of the indicated proteins before or after treatment with 100nM BEZ235, BAY236, BAY439 or Rapamycin (Rapa). Supplementary Figure 5 Knockdown efficiency of FOXO3a in isogenic in vitro models. Immunoblot analysis to determine the effectiveness of each of our FOXO3a knockdown constructs.
4 Supplementary Figure 6 TRIB2 confers resistance to 5-Flurouracil. Matched isogenic TRIB2 cell line FACS analysis following 72 hour exposure to 5-Flurouracil (n=6). P values are indicated for each comparison by two-way analysis of variance (ANOVA) and data represent the mean ± standard deviation (s.d.).
5 Supplementary Figure 7 TRIB2 negatively regulates FOXO3a by the COP1 domain. a, Schematic of TRIB2 constructs used in our transient transfection experiments. b, qrt-pcr analysis of p27 and FasLG expression in our 293T cell line 24 hours post transient transfection of each indicated construct (n=6). * (P 0.05) are indicated for each comparison by two-way ANOVA and data represent the mean ± s.d. c, (left) Immunoblot analysis for p27 or FasLG expression following the transient transfection of each TRIB2 construct shown in a. (right) Quantification of p27 or FasLG protein expression for 293T cells before and 24 hours post BEZ235 exposure. Data represent the mean ± s.d.
6 Supplementary Figure 8 TRIB2 protein expression in ex vivo primary colon or pancreatic cancer prior to first line chemotherapy. a, Immunoblot analysis for TRIB2 in matched colon cancer patient sample (n = 14). b, TRIB2 immunoblot analysis from pancreatic cancer patients following surgical removal (n = 7). Supplementary Figure 9 TRIB1 and TRIB3 are not differentially expressed in metastatic melanoma clinical samples. qrt-pcr analysis of TRIB1 and TRIB3 expression in ex vivo metastatic melanoma samples (n = 20) versus normal control tissue (n = 10). Data were analysed by two-way ANOVA and values represent the mean ± s.d.
7 Supplementary Figure 10 TRIB2, pser473-akt and pser253-foxo3a protein expression correlates with clinical prognosis for melanoma patients. a. Average protein quantification of normal
8 and melanoma patient tissue samples separated based on clinical outcome (CR, complete response, SD, stable disease, PD, progressive disease) presented in figure 4d. Samples between clinical cohorts were normalized to β-actin and relative intensity measured by ImageJ. Data is presented +/- St.Dev. and P values indicated by two-tailed t-test. b-l. Individual protein expression for every sample within each patient cohort. The average expression is indicated by the red line shown in each group. Supplementary Figure 11 High TRIB2 expression correlates with significantly worse clinical prognosis for colon cancer patients. a, Kaplan-Meier analysis of colon cancer patients (n = 177) classified based on low ( 1.5 fold TRIB2 expression) or high ( 2.5 fold TRIB2 expression) 25. Patients with low TRIB2 expression improved patient survival (log-rank P = ). b, Kaplan-Meier analysis of pancreatic cancer patients (n = 38) classified based on low ( 1.5 fold TRIB2 expression) or high ( 2.5 fold TRIB2 expression) 26. Low TRIB2 expression displayed a trend towards, although this was not a statistically significant increase in overall patient survival (log-rank P = 0.205).
9 Supplementary Figure 12 TRIB1 or TRIB3 expression does not correlate with clinical prognosis for melanoma, colon or pancreatic cancer patients. a, Kaplan-Meier analysis of melanoma patients (n = 38) classified based on low ( 1.5 fold TRIB1 expression) or high ( 2.5 fold TRIB1 expression). Low TRIB1 expression did not improve patient survival (log-rank P = 0.656). b, Kaplan-Meier analysis of melanoma patients (n = 38) classified based on low ( 1.5 fold TRIB3 expression) or high ( 2.5 fold TRIB3 expression). Low TRIB3 expression did not improve patient survival (log-rank P = 0.079). c, Kaplan-Meier analysis of colon cancer patients (n = 177) classified based on low ( 1.5 fold TRIB1 expression) or high ( 2.5 fold TRIB1 expression) 25. TRIB1 expression did not affect patient survival (logrank P = 0.667). d, Kaplan-Meier analysis of colon cancer patients (n = 177) classified based on low ( 1.5 fold TRIB3 expression) or high ( 2.5 fold TRIB3 expression) 25. TRIB3 expression did not affect patient survival (log-rank P = 0.492). e, Kaplan-Meier analysis of pancreatic cancer patients (n = 38) classified based on low ( 1.5 fold TRIB1 expression) or high ( 2.5 fold TRIB1 expression) 26. TRIB1 expression had no statistically significant effect on survival (log-rank P = 0.143). f, Kaplan-Meier analysis of pancreatic cancer patients (n = 38) classified based on low ( 1.5 fold TRIB3 expression) or high ( 2.5 fold TRIB3 expression) 26. TRIB3 expression had no statistically significant effect on patient survival (log-rank P = 0.973).
10 Supplementary Table 1 Cell lines used in this study. Cell line 293T Empty 293T +TRIB2 U2OS Empty U2OS +TRIB2 G361 TRIB2 G361 +TRIB2sh SK-Mel28 TRIB2 SK-Mel28 +TRIB2sh 293T empty/foxo3akd 293T +TRIB2/FOXO3aKD U2OS empty/foxo3akd U2OS +TRIB2/FOXO3aKD Parental cell line (supplier) HEK293T (ATCC) HEK293T (ATCC) U2OS (ATCC) U2OS (ATCC) G361 (ATCC) G361 (ATCC) SK-Mel28 (ATCC) SK-Mel28 (ATCC) HEK293T (ATCC) HEK293T (ATCC) U2OS (ATCC) U2OS (ATCC) Origin Manipulation TRIB2 level FOXO3a level Embryonal kidney Embryonal kidney GFP over expression Low (endogenous) Endogenous TRIB2-GFP over expression High (endogenous + exogenous) Endogenous Osteosarcoma GFP over expression Low (endogenous) Endogenous Osteosarcoma TRIB2-GFP over expression High (endogenous + exogenous) Endogenous Melanoma Scramble shrna sequence High (endogenous) Endogenous Melanoma TRIB2 shrna sequence Low > 90% reduction compared to control Endogenous Melanoma Scramble shrna sequence High (endogenous) Endogenous Melanoma Embryonal kidney Embryonal kidney Osteosarcoma Osteosarcoma TRIB2 shrna sequence GFP over expression stable FOXO3a shrna expression TRIB2-GFP over expression, stable FOXO3a shrna expression GFP over expression, stable FOXO3a shrna expression TRIB2-GFP over expression, stable FOXO3a shrna expression Low> 90% reduction compared to control Low (endogenous) High (endogenous + exogenous) Low (endogenous) High (endogenous + exogenous) Endogenous > 80% reduction compared to control > 80% reduction compared to control. > 80% reduction compared to control > 80% reduction compared to control
11 Supplementary Table 2 Antibodies used for immunoblotting in this study. Antibody Species Supplier Ratio used Tribbles2 (TRIB2) Rabbit Custom raised CNIO Madrid 1:1000 caspase-3 (H-277) Rabbit Santa Cruz Biotechnology (SCBT) 1:250 β actin (C4) Mouse SCBT 1:2000 Akt1 (C-20) Goat SCBT 1:1000 pser473-akt1/2/3 Rabbit SCBT 1:1000 PDK1 (C-20) Goat SCBT 1:1000 p70 S6 kinase α (C-18) Rabbit SCBT 1:1000 pthr389-p70 S6 kinase α Goat SCBT 1:500 FKHRL1 (FOXO3a) (N-16) Goat SCBT 1:500 pser253-fkhrl1 (FOXO3a) Rabbit SCBT 1:1000 FAS-LG (C-178) Rabbit SCBT 1:1000 Bim (H-191) Rabbit SCBT 1:1000 p27 (F-8) Mouse SCBT 1:1000 p53 (DO-1) Mouse SCBT 1:1000 MDM2 (C-18) Rabbit SCBT 1:1000 pser166-mdm2 Rabbit Cell Signalling Technology (CST) 1:1000 pser241-pdk1 Rabbit CST 1:1000 pser792-raptor Rabbit CST 1:1000
12 Supplementary Table 3 Quantitative real time PCR primers used in this study. Gene 5-3 Fwd 5-3 Rev CDKN1B (p27) CCTCCTCCAAGACAAACAGC CTGTATTTGGAGGCACAGCA BCL2-like 11 (BIM) GCCCCTACCTCCCTACAGAC AAGATGAAAAGCGGGGATCT FasLG TCTTCCCTGTCCAACCTCTG TGCTGTGGTTCCCTCTCTTC CDKN2A (p16) CTTCCTGGACACGCTGGT GCATGGTTACTGCCTCTGGT TRIBBLES1 (TRIB1) AAGGGGGAAGATGATGCTTT TGTGCTCAGGAATGCAGAAC TRIBBLE2 (TRIB2) CACAGGTCTACCCCCATCAC CCCGATACAAGAAACGCAAT TRIBBLES3 (TRIB3) AGGAAGAAGCGGTTGGAGTT TGCACGATCTGGAGCAGTAG CDKN1A (p21) GACACCACTGGAGGGTGACT CTGCCTCCTCCCAACTCAT TRAIL ACCAACGAGCTGAAGCAGAT ACGGAGTTGCCACTTGACTT AKT1 AGAAGCAGGAGGAGGAGGAG CCCAGCAGCTTCAGGTACTC Homo sapiens MDM2 CAGCTTCGGAACAAGAGACC GTCCGATGATTCCTGCTGAT GAPDH CAATGACCCCTTCATTGACC TTGATTTTGGAGGGATCTCG Supplementary Table 4 shrna sequences used in this study. Gene Plasmid backbone Sequence FOXO3a #1 pretrosuper GCAGGCCTCATCTCAGAGCTCTCTTGAAGCTCTGAGATGAGGCCTGC FOXO3a #2 pretrosuper CTGCGACGGCTGACTGAAATCTCTTGAATTTCAGTCAGCAGTCGCAG FOXO3a #3 pretrosuper CCTGATGGGGGAAANANCTCTCTTGAANCTCTGANATGANGCCTGC FOXO3a #4 pretrosuper CGTGACTGTCGCAATGATCTCTTGAAGATCAGTGCGACAGTCACG TRIBBLE2 (TRIB2) #1 pretrosuper CCAGGACGAGATCCAGTTCTCTTGGATCCAA TRIBBLE2 (TRIB2) #2 pretrosuper CCTGCCGACCGAGGACCCCCGTTGGATCCAA TRIBBLE2 (TRIB2) #3 pretrosuper TAAAATCAGTGGCACCGAGGCTTGGATCCAA TRIBBLE2 (TRIB2) #4 pretrosuper CTCCCTCTTCAGCAAGATCCGTTGGATCCAA
13 Supplementary Table 5 CEBPα and CEBPα-dependent gene expression analysis. Gene locus log 2 (fold change) P value q value Sig? Fold down regulation. Reference CEBPα chr19: Yes SOX4 chr6: Yes TGFBR2 chr3: Yes CD33 chr19: Yes SLC2A4 chr17: Yes UCA1 chr19: Yes Zhang, H., et al. (2013). Cancer Cell 24(5): Takayama, K., et al. (2014). Development 141(1): Shamsasenjan, K., et al. (2009). Int J Hematol 89(3): Fujimoto,M., et al. (2005). Biochimica et Biophysica Acta (BBA) 1745(1): Xue, M., et al. (2014). Oncol Rep 31(5): Supplementary Table 6 Multivariate analysis of TRIB2 expression and clinical confounding factors (GSE65904). Variables in the model: TRIB2 expression, stage, age and gender. Parameter P value HR 95% C (lower) 95% Cl (upper) Stage General In transit Local Primary Regional TRIB2 High vs Low Supplementary Table 7 Multivariate analysis of TRIB2 expression and clinical confounding factors (GSE65904). Variables in the model: TRIB2 expression, stage, age and gender. Parameter P value HR 95% C (lower) 95% Cl (upper) Age 60 vs < Gender Male vs Female < TRIB2 High vs Low
14 Supplementary Figure 13 Uncropped scans of western blots presented in Figure 1b.
15 Supplementary Figure 14 Uncropped scans of western blots presented in Figure 2a
16 Supplementary Figure 15 Uncropped scans of western blots presented in Figure 2b
17 Supplementary Figure 16 Uncropped scans of western blots presented in Figure 2c
18 Supplementary Figure 17 Uncropped scans of western blots presented in Figure 2e
19 Supplementary Figure 18 Uncropped scans of western blots presented in Figure 3d
20 Supplementary Figure 19 Uncropped scans of western blots presented in Figure 3f
21 Supplementary Figure 20 Uncropped scans of western blots presented in Figure 3h
22 Supplementary Figure 21 Uncropped scans of western blots presented in Figure 4c
23 Supplementary Figure 22 Uncropped scans of western blots presented in Figure 4e
24 Supplementary Figure 23 Uncropped scans of western blots presented in Supplementary Figure 1
25 Supplementary Figure 24 Uncropped scans of western blots presented in Supplementary Figure 4
26 Supplementary Figure 25 Uncropped scans of western blots presented in Supplementary Figure 5
27 Supplementary Figure 26 Uncropped scans of western blots presented in Supplementary Figure 7
28 Supplementary Figure 27 Uncropped scans of western blots presented in Supplementary Figure 8
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Supplementary Figure 1. DJ-1 modulates ROS concentration in mouse skeletal muscle. (a) mrna levels of Dj1 measured by quantitative RT-PCR in soleus, gastrocnemius (Gastroc.) and extensor digitorum longus
Supplemental Information. NRF2 Is a Major Target of ARF. in p53-independent Tumor Suppression
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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
Normal Skin. Tissue Samples and Melanoma Cell Lines. BRAF Mut. RAS Mut RAS WT /BRAF WT
Supplemental Figure 1. MERTK gene expression in melanoma cell line panel from Cancer Cell Line Encyclopedia. A. Microarray analysis of melanoma cell lines from UNC collection grouped by oncogenic mutation.
GPR120 *** * * Liver BAT iwat ewat mwat Ileum Colon. UCP1 mrna ***
a GPR120 GPR120 mrna/ppia mrna Arbitrary Units 150 100 50 Liver BAT iwat ewat mwat Ileum Colon b UCP1 mrna Fold induction 20 15 10 5 - camp camp SB202190 - - - H89 - - - - - GW7647 Supplementary Figure
Supplemental Figure 1
Supplemental Figure 1 1a 1c PD-1 MFI fold change 6 5 4 3 2 1 IL-1α IL-2 IL-4 IL-6 IL-1 IL-12 IL-13 IL-15 IL-17 IL-18 IL-21 IL-23 IFN-α Mut Human PD-1 promoter SBE-D 5 -GTCTG- -1.2kb SBE-P -CAGAC- -1.kb
Figures S1-S5, Figure Legends, Table S1 List of primers used in the study
Insulin receptor alternative splicing is regulated by insulin signaling and modulates beta cell survival Pushkar Malakar,4, Lital Chartarifsky,4, Ayat Hija, Gil Leibowitz 3, Benjamin Glaser 3, Yuval Dor,
Supplementary Figure 1
Supplementary Figure 1 a γ-h2ax MDC1 RNF8 FK2 BRCA1 U2OS Cells sgrna-1 ** 60 sgrna 40 20 0 % positive Cells (>5 foci per cell) b ** 80 sgrna sgrna γ-h2ax MDC1 γ-h2ax RNF8 FK2 MDC1 BRCA1 RNF8 FK2 BRCA1
Supplementary Fig. 1. Delivery of mirnas via Red Fluorescent Protein.
prfp-vector RFP Exon1 Intron RFP Exon2 prfp-mir-124 mir-93/124 RFP Exon1 Intron RFP Exon2 Untransfected prfp-vector prfp-mir-93 prfp-mir-124 Supplementary Fig. 1. Delivery of mirnas via Red Fluorescent
Supplementary Figure 1. SA-β-Gal positive senescent cells in various cancer tissues. Representative frozen sections of breast, thyroid, colon and
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Supplementary Figure 1. AdipoR1 silencing and overexpression controls. (a) Representative blots (upper and lower panels) showing the AdipoR1 protein
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Supplemental Information. Menin Deficiency Leads to Depressive-like. Behaviors in Mice by Modulating. Astrocyte-Mediated Neuroinflammation
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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
Supplementary Table 1. List of primers used in this study
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7SK ChIRP-seq is specifically RNA dependent and conserved between mice and humans.
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Supplementary Figure 1. Characterization of NMuMG-ErbB2 and NIC breast cancer cells expressing shrnas targeting LPP. NMuMG-ErbB2 cells (a) and NIC
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Supplementary Figure 1. Expression of CUGBP1 in non-parenchymal liver cells treated with TGF-β
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SUPPLEMENTARY FIGURES AND TABLE
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GW(g)/BW(g) GW(g)/BW(g) Con Dex Con Dex. GW(g)/BW(g) Relative mrna levels. Atrogin-1 Murf-1. Atrogin-1 Murf-1. Soleus
a b c GW(g) GW(g) GW(g) 0.20 0.15 0.10 0.05 0.00 0.20 0.15 0.10 0.05 Den * ** GW(g)/BW(g) Den Dex Dex GW(g)/BW(g) d 0.00 Fasting GW(g) e 0.15 Cancer Cachexia f GW(g) 0.10 0.05 0.00 Young ** Old g GW(g)/BW(g)
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Supplementary Figure 1. HeliScope CAGE revealed androgen-regulated signaling and differentially regulated promoters in hormone-refractory prostate
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SUPPLEMENTARY INFORMATION
doi: 10.1038/nature05732 SUPPLEMENTARY INFORMATION Supplemental Data Supplement Figure Legends Figure S1. RIG-I 2CARD undergo robust ubiquitination a, (top) At 48 h posttransfection with a GST, GST-RIG-I-2CARD
Nature Medicine: doi: /nm.4324
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 Supplementary Figure 1. Kinetics of SnCs development in surgically-induced OA and effect of GCV-induced SnC clearance on OA disease progression
SUPPLEMENTARY INFORMATION
DOI:.38/ncb3399 a b c d FSP DAPI 5mm mm 5mm 5mm e Correspond to melanoma in-situ Figure a DCT FSP- f MITF mm mm MlanaA melanoma in-situ DCT 5mm FSP- mm mm mm mm mm g melanoma in-situ MITF MlanaA mm mm
1.5 ASK1KO fed. fasted 16 hrs w/o water. Fed. 4th. 4th WT ASK1KO N=29, 11(WT), ,5(ASK1KO) ASK1KO ASK1KO **** Time [h]
![1.5 ASK1KO fed. fasted 16 hrs w/o water. Fed. 4th. 4th WT ASK1KO N=29, 11(WT), ,5(ASK1KO) ASK1KO ASK1KO **** Time [h]](/thumbs/87/97258189.jpg "1.5 ASK1KO fed. fasted 16 hrs w/o water. Fed. 4th. 4th WT ASK1KO N=29, 11(WT), ,5(ASK1KO) ASK1KO ASK1KO **** Time [h]")
7: 13: 19: 1: 7: 151117 a 151117 4th 4th b c RQ.95 KO.9.85.8.75.7 light dark light dark.65 7: 19: 7: 19: 7: Means ± SEM, N=6 RQ 1..9.8.7.6.6 KO CL (-) CL (+) ibat weight ratio (/body weight) [%].5.4.3.2.1
Dox. R26-rtTA Tyr-CreERT2. any ink/arf, no rtta (n=8) ink/arf +/+ (n=5) Day 0 Day 11 Day 18 Day 28
A 4OHT Dox hraf iip tumors inras ddh 2 O -RT Ink/Arf / Pten l/ l R26-lsl-rtTA Tyr-reERT2 TetO-hRAF V6E Ink/Arf / Pten / R26-rtTA Tyr-reERT2 TetO-hRAF V6E Ink/Arf / Pten / R26-rtTA Tyr-reERT2 TetO-hRAF
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
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Supplementary Figure 1. Normal T lymphocyte populations in Dapk -/- mice. (a) Normal thymic development in Dapk -/- mice. Thymocytes from WT and Dapk
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Supplementary Discussion The cell cycle machinery and the DNA damage response network are highly interconnected and co-regulated in assuring faithful duplication and partition of genetic materials into
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Supplementary methods:
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ACC ELOVL MCAD. CPT1α 1.5 *** 0.5. Reverbα *** *** 0.5. Fasted. Refed
Supplementary Figure A 8 SREBPc 6 5 FASN ELOVL6.5.5.5 ACC.5.5 CLOCK.5.5 CRY.5.5 PPARα.5.5 ACSL CPTα.5.5.5.5 MCAD.5.5 PEPCK.5.5 G6Pase 5.5.5.5 BMAL.5.5 Reverbα.5.5 Reverbβ.5.5 PER.5.5 PER B Fasted Refed
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