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1 Supplemental Information Tobacco-specific Carcinogen Induces DNA Methyltransferases 1 Accumulation through AKT/GSK3β/βTrCP/hnRNP-U in Mice and Lung Cancer patients Ruo-Kai Lin, 1 Yi-Shuan Hsieh, 2 Pinpin Lin, 3 Han-Shui Hsu, 4 Chih-Yi Chen, 5 Yen-An Tang 1, Chung-Fan Lee, 6 and Yi-Ching Wang 1,* 1
2 Supplemental figures and legends A549 H1299 (bp) MW DMSO NNK DMSO NNK β-actin DNMT1 Supplemental Figure 1 NNK-treated A549 and H1299 cells showed no change in the endogenous DNMTs mrna expression levels. The expression level was measured by RT-PCR The housekeeping gene β- actin was used as an internal control. 2
3 Supplemental Figure 2 The signaling pathways induced by NNK. (A) IMR90 normal lung cell line and A549 lung cancer cell lines were treated with 10 µm NNK for various times as indicated. Phosphorylation of AKT was analyzed by Western blotting using phospho-akt at Ser473 antibody. Total AKT antibody was used to confirm and quantify AKT protein. Western blot analysis was performed to detect phosphorylated NFκB or total NFκB by using a phospho-specific NFκB or total NFκB antibodies; phosphorylated ERK1/2 or total ERK1/2 by using a phospho-specific ERK1/2 or a mixture of ERK1 and ERK2 antibodies. Phosphorylation of p38 was analyzed by Western blotting using phospho-p38 antibody. β-actin used an internal control. (B) Quantitative figures were determined from at least three experiments. Columns, mean; bars, ±SEM. and P values are as indicated. 3
4 Supplemental Figure 3 GSK3β involved in DNMT1 protein degradation and the increase of DNMT1 protein level by NNK in IMR90 cells. (A) IMR90 cells were treated with or without 25 µm GSK3β inhibitor, SB for 24 hr, and then treated with 10 µm NNK for 2 hr. Increased in DNMT1 protein expression after inhibition of GSK3β activity by NNK and SB was observed (lanes 2-4 versus lane 1 in DNMT1 level). Phosphorylated GSK3β ser9 (inactive form can be seen after NNK and SB treatment (lanes 2-4 versus lane 1 in GSK3β ser9 level). Protein expression levels of DNMT1, phosphorylation of GSK3β ser9, and total GSK3β were analyzed by Western blotting with β-actin as an internal control. Densitometry showing the change of DNMT1 protein level. (B) Knockdown of GSK3β in cells treated by sirna enhanced DNMT1 protein level. Columns, mean; bars, ±SEM (n=3). P values are as indicated. 4
5 Supplemental Figure 4 Exogenous GSK3β and βtrcp induced DNMT1 protein degradation. (A) GSK3β and (B) βtrcp promoted DNMT1 protein degradation in a dosedependent manner as seen using both Western blot and densitometry assays. A549 cells were transfected with pcmv-sport6-gsk3β or pcmv-sport6- βtrcp at indicated doses for 24 h. Columns, mean; bars, ±SEM (n=3). P values are as indicated. 5
6 Supplemental Figure 5 An increase of DNMT1 and p-gsk3β Ser9 expression and their co-localization (yellow color) were observed in cells treated with NNK. Immunofluorescence staining of DNMT1 (green color) and p-gsk3βser9 (red color) was performed and visualized by confocal microscopy. Scale bar: 40 _m. 6
7 Supplemental Figure 6 The N-terminal domain of DNMT1 could be induced by NNK treatment which was important for βtrcp interaction. (A) Analysis of two cdna deletion constructs of His-tag DNMT1 expression vector, which contained either amino acids or of DNMT1, are shown. The N-terminal domain of amino acids could be induced by NNK treatment. (B) Immunoprecipitation assay was performed in A549 cells transfected with control vector or pcmv-sport6-βtrcp with various regions for 24 hr. Cell lysates were immunoprecipitated with anti-βtrcp antibody and Western blotted with anti-his-dnmt1 antibody. Normal-IgG is a negative control. Data demonstrated that N-terminal domain of amino acids of DNMT1 was important for βtrcp interaction. 7
8 Supplemental Figure 7 NNK-induced DNMT1 accumulation by AKT/GSK3β/βTrCP/hnRNP-U signaling pathway was not due to the impact of NNK treatment on the cell cycle. (A) Flow cytometry showed that 10µM of NNK treatment for 24 hours did not change cell cycle distribution in A549 cells (upper panel). (B) Flow cytometry was performed in A549 cells treated with aphidocolin, nocodazole, and 5 hour post-release from nocodazole to confirm the induction of S, G2/M, and G1 phases, respectively. Western or Immunoprecipitation assay (lower panel) of cell treated with 10 µm NNK at specific cell cycle phases to show the induction of DNMT1 protein level and the interaction between DNMT1 and hnrnpu, βtrcp, and GSK3β. Data demonstrated that NNK-induced DNMT1 accumulation could be seen in all phases of cell cycle. 8
9 Supplemental Tables Supplemental Table 1 Multivariate Analysis of factors associated with poor survival A. Variable Risk Ratio 95%CI P Age ( ) Tumor type ( ) Tumor stage ( ) Smoking and DNMT1(+) ( ) A. Results were analyzed by the Cox proportional hazards regression modal. 9
10 Supplemental Table 2 The mrna expression of DNMT1 was not associated with the smoking status of patients A Characteristics DNMT1 mrna Total Non-overexpression Overexpression N N (%) N (%) Overall (51.1) 37 (48.9) Smoking habit Smoker (58.6) 19 (43.2) Non-smoker B (56.1) 18 (43.9) A. Results were analyzed by the Pearson Chi-Square test. Degree of freedom = 1. P value with significance is shown as superscript. B. The non-smoker group includes ex-smoker and never smoker. 10
11 Supplemental Table 3. Overexpression of DNMTs proteins and clinical parameters in NSCLC tumors. A DNMT1 Total B Characteristics High expression Normal expression n n (%) n (%) Overall (48.4) 64 (51.6) Clinicopathological parameters Age < (54.3) 16 (45.7) > (46.1) 48 (53.9) Tumor type SQ (50.0) 24 (50.0) AD (49.3) 34 (50.7) Tumor stage I + II (46.7) 32 (53.3) III + IV (50.0) 32 (50.0) A. Results were analyzed by the Pearson Chi-Square test. Degree of freedom = 1. P value with significance is shown as superscript. B. Total number of samples (n) in some categories is less than the overall number analyzed because clinical data was not available for some samples. 11
12 Supplemental Table 4 The antibodies and their reaction conditions used in the present study Raised Catalog Target K.D. Application Dilution Source In No. Anti-DNMT1 190 Chicken Western blot 1:10000 Immunoprecipitation 1:100 Immunohistochemistry Immunofluorescence 1:500 Asia Hepato Gene Co. A22659 Mouse Immunohistochemistry 1:30 Imgenex IMG261A Anti-_-actin 42 Mouse Western blot 1:5000 Anti-LAMIN A/C 62/69 Mouse Western blot 1:2000 Anti-GAPDH 38 Mouse Western blot 1:2000 Anti-AKT 60 Rabbit Western blot 1:1000 Immunoprecipitation 1:100 Novus Biologicals Santa Cruz Santa Cruz Cell Signaling NB sc-7292 Sc Anti-phospho- AKT Ser Rabbit Western blot 1:500 Cell 9271 Immunohistochemistry 1:30 Signaling 3787 Anti-Erk1/2 42,44 Rabbit Western blot 1:1000 Upstate Anti-phospho- 42/44 Mouse Western blot 1:1000 Upstate Erk1/2 Anti-phosphop38 Thr Rabbit Western blot 1:1000 Chemicon AB3828 Anti-NF_B 65 Rabbit Western blot 1:1000 Upstate Anti-phophor- NF_B Ser Rabbit Western blot 1:1000 Anti-GSK3β 46 Rabbit Western blot 1:5000 Immunoprecipitation 1:125 Western blot 1:1000 Anti-phospho- GSK3β Ser9 46 Rabbit Immunohistochemistry 1:100 Immunofluorescence 1:100 Anti-βTrCP 70 Cell Signaling Cell Signaling Nuvus NB Mouse Western blot 1:500 Zymed Goat Immunohistochemistry Immunofluorescence 1:300 Immunoprecipitation 1:25 Santa Cruz sc-8862 Anti-p53 53 Mouse Western blot 1:1000 DAKO DO-7 12
13 Anti-His -- A Rabbit Western blotting 1:500 Immunoprecipitation 1:50 Cell Signaling 2365 Anti-phospho-serine -- A Mouse Western blotting 1:500 Immunoprecipitation 1:50 Sigma P3430 Anti-hnRNP-U 130 Mouse Western blotting 1:2000 Immunofluorescence Immunoprecipitation 1:50 Santa Cruz sc Anti-hnRNP-U 130 Rabbit Immunofluorescence Immunohistochemistry 1:50 Santa Cruz sc Anti-ubiquitin -- A Mouse A. Western blotting 1:1000 Immunoprecipitation 1:50 Santa Cruz sc Molecular weight is variable. 13
14 Supplemental Table 5 List of primers and sirna sequences and their reaction conditions used in the present study Gene A primer 5 3 sequences PCR size Tm Cycle DNMT1-RT-PCR _-actin-rt-pcr p16-bis-sequencing p16-methyl-msp-o p16-methyl-msp-u p16-methyl-msp-m RAR_-methyl-MSP-O RAR_-methyl-MSP-U RAR_-methyl-MSP-M FHIT-methyl-MSP-O ACCGCTTCTACTTCCTCGAGGCCTA GTTGCAGTCCTCTGTGAACACTGTGG ACACTGTGCCCATCTACGAGG AGGGGCCGGACTCGTCATACT GTTTTTTTGTTTGGAAAGATAT CRCCCRCACCTCCTCTACCC GTTTTTTTGTTTGGAAAGATAT CTACCCATCATCATAACCTAAATC TTATTAGAGGGTGGGGTGGATTGT CAACCCCAAACCACAACCATAA TTATTAGAGGGTGGGGCGGATCGC GACCCCCGAACCGCGACCTAA AAGTGAGTTGTTTAGAGGTAGGAGGG CCTATAATTAATCCAAATAATCATTTACC TTGAGAATGTGAGTGATTTGA AACCAATCCAACCAAAACAA TCGAGAACGCGAGCGATTCG GACCAATCCAACCGAAACGA TAGTGGGTATATTTTTAG ATCCCACCCTAAAACCTC o C o C o C o C o C o C o C o C o C o C 40 FHIT-methyl-MSP-U GAAGGTAGGGGTGGGGAGGTAAGTT o C 35 14
15 CATAAACAACACCAACCCCACTA FHIT-methyl-MSP-M GAAGGTAGGGGCGGGGAGGTAAGTT CGTAAACGACGCCAACCCACTA o C 35 p16-promoter-chip TTGAAGCTGGTCTTTGGAT ATCGAAATCACCTGTACGACT o C 35 RAR_-promoter-ChIP GGCAACCAACATGGCTATTT TATAGTGGTCAAGAGCATGGGT o C 35 FHIT-promoter-ChIP ATGCTCTGCGCGTATTGGC GGTGATGACCTGACGCG o C 35 GAPDH-promoter-ChIP AATGAAAGGCACACTGTCTCTCTC GTTTCTGCACGGAAGGTCAC o C 35 RNAi of AKT AUUCUUGAGGAGGAAGUAGCGUGGC GCCACGCUACUUCCUCCUCAAGAAU RNAi of GSK3β AUUACUUGACAGUUCUUGAGUGGUG CACCACUCAAGAACUGUCAAGUAAU A RT-PCR: reverse-transcriptase polymerase chain reaction; MSP: methylation-specific PCR; O: outer primer; U: unmethylation - specific primer; M: methylation-specific primer; ChIP: chromatin immunoprecipitation; RNAi: interference RNA. 15
Institute of Molecular Medicine, College of Medicine, National Taiwan University, Taipei, Republic of China.
Research article The tobacco-specific carcinogen NNK induces DNA methyltransferase 1 accumulation and tumor suppressor gene hypermethylation in mice and lung cancer patients Ruo-Kai Lin, 1 Yi-Shuan Hsieh,
(a) Significant biological processes (upper panel) and disease biomarkers (lower panel)
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SUPPLEMENTARY INFORMATION. Supplementary Figures S1-S9. Supplementary Methods
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supplementary information
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Supplementary Figures
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Supplementary Information
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Supplementary Information Titles Journal: Nature Medicine
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Supporting Information
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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 1
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Supplementary Figure 1.
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SUPPLEMENTARY FIGURES
SUPPLEMENTARY FIGURES Figure S1. Clinical significance of ZNF322A overexpression in Caucasian lung cancer patients. (A) Representative immunohistochemistry images of ZNF322A protein expression in tissue
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1 a b Supplementary Figure 1. Effects of GSK3b knockdown on poly I:C-induced cytokine production. RAW264.7 cells stably expressing control shrna (Con) or GSK3b-specific shrna (sh- GSK3b) were stimulated
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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
Figures S1-S5, Figure Legends, Table S1 List of primers used in the study
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SUPPLEMENTARY FIGURE LEGENDS. atypical adenomatous hyperplasias (AAH); Grade II: adenomas; Grade III: adenocarcinomas;
SUPPLEMENTARY FIGURE LEGENDS Supplementary Figure S1: Tumor grades in Ras G12D ; p53 / lung tumors. Representative histology (H&E) of K-Ras G12D ; p53 / lung tumors 13 weeks after tumor initiation. Grade
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
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Shao-Ming Shen et al Role of I in MT of cancers MO reports xpanded View igures igure V1. nalysis of the expression of I isoforms in cancer cells and their interaction with PTN. RT PR detection of Ish and
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RNA extraction, RT-PCR and real-time PCR. Total RNA were extracted using
Supplementary Information Materials and Methods RNA extraction, RT-PCR and real-time PCR. Total RNA were extracted using Trizol reagent (Invitrogen,Carlsbad, CA) according to the manufacturer's instructions.
Supporting Information. Supporting Tables. S-Table 1 Primer pairs for RT-PCR. Product size. Gene Primer pairs
Supporting Information Supporting Tables S-Table 1 Primer pairs for RT-PCR. Gene Primer pairs Product size (bp) FAS F: 5 TCTTGGAAGCGATGGGTA 3 429 R: 5 GGGATGTATCATTCTTGGAC 3 SREBP-1c F: 5 CGCTACCGTTCCTCTATCA
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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
CYLD Negatively Regulates Transforming Growth Factor-β Signaling via Deubiquitinating Akt
Supplementary Information CYLD Negatively Regulates Transforming Growth Factor-β Signaling via Deubiquitinating Akt Jae Hyang Lim, Hirofumi Jono, Kensei Komatsu, Chang-Hoon Woo, Jiyun Lee, Masanori Miyata,
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Supplementary Figures Supplementary Figure S1. Binding of full-length OGT and deletion mutants to PIP strips (Echelon Biosciences). Supplementary Figure S2. Binding of the OGT (919-1036) fragments with
MicroRNAs Modulate the Noncanonical NF- B Pathway by Regulating IKK Expression During Macrophage Differentiation
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Name Animal source Vendor Cat # Dilutions
Supplementary data Table S1. Primary and Secondary antibody sources Devi et al, TXNIP in mitophagy A. Primary Antibodies Name Animal source Vendor Cat # Dilutions 1. TXNIP mouse MBL KO205-2 1:2000 (WB)
Fang et al. NMuMG. PyVmT unstained Anti-CCR2-PE MDA-MB MCF MCF10A
A NMuMG PyVmT 16.5+.5 47.+7.2 Fang et al. unstained Anti-CCR2-PE 4T1 Control 37.6+6.3 56.1+.65 MCF1A 16.1+3. MCF-7 3.1+5.4 MDA-M-231 42.1+5.5 unstained Secondary antibody only Anti-CCR2 SUPPLEMENTAL FIGURE
Figure 1. Possible role of oncogene activation, receptor, G-protein mutation, or tumor
Figures Part of introduction Figure 1. Possible role of oncogene activation, receptor, G-protein mutation, or tumor supressor gene deletion in the induction of thyroid carcinoma. ( by James A Fagin, M.D.)
Supplementary Table 1. Metabolic parameters in GFP and OGT-treated mice
Supplementary Table 1. Metabolic parameters in GFP and OGT-treated mice Fasted Refed GFP OGT GFP OGT Liver G6P (mmol/g) 0.03±0.01 0.04±0.02 0.60±0.04 0.42±0.10 A TGs (mg/g of liver) 20.08±5.17 16.29±0.8
Supplementary Materials for
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P-Akt Thr308. T-Akt *** *** Anti-α3 IgG Ctrl
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Supplementary material. Supplementary Figure legends
Supplementary material Supplementary Figure legends Supplementary Figure 1: Senescence-associated proliferation stop in response to oncogenic N-RAS expression Proliferation of NHEM cells without (ctrl.)
Downregulation of the small GTPase SAR1A: a key event underlying alcohol-induced Golgi fragmentation in hepatocytes
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Supplementary Fig. 1: ATM is phosphorylated in HER2 breast cancer cell lines. (A) ATM is phosphorylated in SKBR3 cells depending on ATM and HER2
Supplementary Fig. 1: ATM is phosphorylated in HER2 breast cancer cell lines. (A) ATM is phosphorylated in SKBR3 cells depending on ATM and HER2 activity. Upper panel: Representative histograms for FACS
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
Supplementary Figure 1. Confocal immunofluorescence showing mitochondrial translocation of Drp1. Cardiomyocytes treated with H 2 O 2 were prestained
Supplementary Figure 1. Confocal immunofluorescence showing mitochondrial translocation of Drp1. Cardiomyocytes treated with H 2 O 2 were prestained with MitoTracker (red), then were immunostained with
Supplementary Figure 1
Supplementary Figure 1 Constitutive EGFR signaling does not activate canonical EGFR signals (a) U251EGFRInd cells with or without tetracycline exposure (24h, 1µg/ml) were treated with EGF for 15 minutes
Supplementary data Supplementary Figure 1 Supplementary Figure 2
Supplementary data Supplementary Figure 1 SPHK1 sirna increases RANKL-induced osteoclastogenesis in RAW264.7 cell culture. (A) RAW264.7 cells were transfected with oligocassettes containing SPHK1 sirna
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
Supplementary Table S1. Tumor samples used for analysis Tumor size (cm) BNG (grade) ERα PR. pn-
Supplementary Table S1. Tumor samples used for analysis Sample# Age Tumor size (cm) pn- Stage Stage BNG (grade) ERα PR HER2 (FISH) Triple negative T1 46 3 N1a III 2 Pos Neg N T2 58 1 N(i-) I 3 Pos Neg
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:
Effects of UBL5 knockdown on cell cycle distribution and sister chromatid cohesion
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HEK293FT cells were transiently transfected with reporters, N3-ICD construct and
Supplementary Information Luciferase reporter assay HEK293FT cells were transiently transfected with reporters, N3-ICD construct and increased amounts of wild type or kinase inactive EGFR. Transfections
Expanded View Figures
MO reports PR3 dephosphorylates TZ Xian-o Lv et al xpanded View igures igure V1. PR3 dephosphorylates and inactivates YP/TZ., Overexpression of tight junction proteins Pals1 () or LIN7 () has no effect
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Supplementary Table 1. Cell sphingolipids and S1P bound to endogenous TRAF2. Sphingolipid Cell pmol/mg TRAF2 immunoprecipitate pmol/mg Sphingomyelin 4200 ± 250 Not detected Monohexosylceramide 311 ± 18
Supplementary Materials for
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DOI: 10.1038/ncb2566 Figure S1 CDKL5 protein expression pattern and localization in mouse brain. (a) Multiple-tissue western blot from a postnatal day (P) 21 mouse probed with an antibody against CDKL5.
Tel: ; Fax: ;
Tel.: +98 216 696 9291; Fax: +98 216 696 9291; E-mail: mrasadeghi@pasteur.ac.ir Tel: +98 916 113 7679; Fax: +98 613 333 6380; E-mail: abakhshi_e@ajums.ac.ir A Soluble Chromatin-bound MOI 0 1 5 0 1 5 HDAC2
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