Transcription Factor Response Elements on Tip: A Sensitive

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TFRE on Tip (TOT): A sensitive approach for large-scale endogenous transcription factor quantitative identification Wenhao Shi, Kai Li, Lei Song, Mingwei Liu, Yunzhi Wang, Wanlin Liu, Xia Xia, Zhao-yu Qin, Bei Zhen, Yi Wang, Fuchu He, Jun Qin, and Chen Ding Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b03150 • Publication Date (Web): 09 Nov 2016 Downloaded from http://pubs.acs.org on November 10, 2016

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Analytical Chemistry

Fudan University Ding, Chen; State Key Laboratory of Proteomics, Beijing Proteome Research Center, Beijing Institute of Radiation Medicine; National Center for Protein Sciences (The PHOENIX center, Beijing); State Key Laboratory of Genetic Engineering and Collaborative Innovation Center for Genetics and Development, School of Life Sciences, Institutes of Biomedical Sciences, Fudan University

ACS Paragon Plus Environment


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TFRE on Tip (TOT): A sensitive approach for large-scale endogenous transcription factor quantitative identification Wenhao Shi1, 2*, Kai Li2, 5*, Lei Song1, 2, Mingwei Liu2, Yunzhi Wang3, Wanlin Liu2, Xia Xia2, Zhaoyu Qin3, Bei Zhen2, Yi Wang4, Fuchu He1, 2, 3#, Jun Qin2, 3,4#, and Chen Ding2, 3#

1

School of Life Sciences, Tsinghua University, Beijing 100084, China

2

State Key Laboratory of Proteomics, Beijing Proteome Research Center, Beijing

Institute of Radiation Medicine; National Center for Protein Sciences (The PHOENIX center, Beijing), Beijing 102206, China 3

State Key Laboratory of Genetic Engineering and Collaborative Innovation Center for

Genetics and Development, School of Life Sciences, Institutes of Biomedical Sciences, Fudan University, Shanghai 200032, China 4

Alkek Center for Molecular Discovery, Verna and Marrs McLean Department of

Biochemistry and Molecular Biology, Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030, USA. 5

Department of pathogeny biology, School of Basic Medical Sciences, North China

University of Science and Technology, Tangshan 063009, Hebei, China

1


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*Equal contributing authors #

To whom correspondence should be addressed: [email protected] (C.D.);

[email protected] (J.Q.); [email protected] (F.H.)

Abstract The ability to map endogenous transcription factors (TFs) DNA binding activity at the proteome scale will greatly enhance our understanding of various biological processes. Here we report a highly sensitive, rapid and high throughput approach, TOT-MS, that allows for quantitative measurement of endogenous TFs. One hundred fifty TFs from 1µg of nuclear extracts can be quantified with single shot mass spectrometry detection in 1 hr machine time. Up to 755 TFs, which is comparable to the depth of RNA-seq, were identified by TOT coupled with on-tip small size reverse-phase liquid chromatography. We further demonstrated the capability of TOT-MS by interrogating the dynamic change of TFs in EGF signaling pathway. This approach should find broad applications in elucidating TF landscape from limited amount of biological materials.

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Introduction TFs are involved in almost all aspects of biological processes, serving as a driving force in development, differentiation, determining and maintaining the fate of organisms1-5. In the human genome, there are about 1,500 TF coding genes, representing the second largest gene superfamily6. In recent years, many high throughput approaches in genome and transcriptome, such as ChIP-seq and RNA-seq, were employed to portray the steady-state and dynamic changes of TFs7,8. These studies have dissected the global transcriptional network in a large scale. However, as the TFs physically bind to the DNA response elements to induce or repress the expressions of downstream genes9, systematic investigation of TF DNA binding activity calls for high-throughput assays that allow identifying and quantifying TFs on proteome scale.

We recently developed an approach using concatenated tandem array of the consensus TFREs (catTFRE) as an affinity reagent to quantitatively analyze endogenous TF DNA binding activities at the proteome scale10. This approach achieved in-depth coverage of TFs at the protein level that is comparable to mRNA level. We further showed that this method could quantitatively measure activated TF changes in response to signaling events. However, this approach requires relatively large amount of sample and 2 days for processing, impeding wider application of the catTFRE in the basic research, industry, and clinical actions that require high 3


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throughput but with limited amount of samples. A more efficient, high throughput and sensitive TF screening approach is in urgent need.

Here we report a miniaturized version of catTFRE - TFRE on Tip (TOT) with high sensitivity, efficiency, throughput that is user friendly. We demonstrate that ~400 TFs from 5µg of nuclear extract (NE) or 150 TFs from as little as 1µg of NE can be identified with about 1 hr of MS running time. Up to 755 TFs, which is comparable to the depth of RNA-seq, were identified by TOT coupled with on-tip small size reverse-phase liquid chromatography. We further demonstrate the feasibility of TOT in screening TFs from micro amount of samples, for example, cells from a single well of 96-well plates, and a piece of clinical gastric endoscope sample. These features of TOT render it suitable for dissecting cellular signaling landscape in basic and clinical research, as well as in drug mechanism screening in industries.

Experimental section Cell culture and tissue samples 293T and HeLa cell lines were purchased from the American Type Culture Collection (Manassas, VA, USA) and cultured in Dulbecco's modified Eagle's medium (DMEM) (Neuronbc 07-02I) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Life Technologies, Inc.), 100U/mL penicillin and 100µg/mL streptomycin in tissue culture flasks at 37°C in 5% CO2. Cells were cultured to 80% confluence on 4



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12-mm cover slips under standard conditions. The media were removed and then washed with PBS, and cells were collected by centrifugation at 1000g for 5 min, washed once with cold PBS and resuspended in cold PBS. For EGF stimulation, HeLa cells were cultured to 70% confluence and starved in minimal essential medium without FBS for 24 h. Then cells were stimulated with 100 ng/ml EGF (sigma 0815AFC05) at 0min, 1min, 20min and 120min, respectively. All experiments were conducted independently in triplicates. Nuclear and cytoplasmic extraction kits (Thermo 78835) with 2-mercaptoethanol were used to prepare NE or WCE from cells, and super-bradford protein assay kit (Cwbio CW0013S) were used to measure protein concentrations. Two cases of endoscopic mucosa samples were also used to extract NE and perform TOT experiments. These two samples were obtained by gastroscopy from a collaborating cooperation hospital. Samples were washed by PBS and then frozen at -80°C until processing. This study was approved by the Ethics Committee of PHOENIX center and performed according to the Declaration of Helsinki Principles.

Preparation of TOT tips. Pipet tips (Axygen TR-222-C) were used to make TOT tips. A 47mm C18 solid phase extraction disk (3M 329574) was inserted to near the end of the tip tail end, then Dynabeads M280 streptavidin (invitrogen 00324960) with TFRE DNA was added into tips (Supplementary figure 1). In general, 20µL Dynabeads M280 (about 5


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1.2-1.4x107 beads) and 0.5pmol catTFRE DNA were used into for each tip. These tips were stored in a refrigerator at 4°C before use. Procedures for TFRE DNA binding to M280 streptavidin were described previously10.

Affinity purification by TOT NE or WCE were added into TOT tips. The tips were laid on ice for 10min, and centrifuged for 5 min at 500g. Tips were then washed with 100µL 50mM NETN (100 mM NaCl, 20 mM Tris-Cl, 0.5 mM EDTA, and 0.5% (vol/vol) Nonidet P-40) twice, and with 100µL PBS solution twice. 0.3-0.5µg trypsin was added on the tips and proteins were digested in 10µL 50mM NH4HCO3 solution for 1h at 37°C. One hundred (100) µL 50% acetonitrile with 0.1% formic acid was used to extract and elute peptides from TOT tips.

LC-MS/MS analysis. Dried peptide samples were re-dissolved in solvent A (0.1% formic acid in water). Liquid chromatography - tandem mass spectrometry (LC-MS/MS) analysis was performed with Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific) equipped with an online Easy-nLC 1000 nano-HPLC system (Thermo Fisher Scientific). The injected peptides were separated on a reverse-phase nano-HPLC C18 column (Pre-column: 3μm, 120 Å, 2 cm × 100μm ID; analytical column: 1.9μm, 120 Å, 10 cm × 100μm ID) at a flow rate of 400nL/min with a 75-min gradient of 5 to 30% 6



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solvent B (0.1% formic acid in acetonitrile). For peptide ionization, 2000 V was applied and a 320°C caplillary temperature was used. For the detection with Oribitrap Fusion mass spectrometry, a precursor scan was measured in the Orbitrap by scanning from m/z 300 –1400 with a resolution of 120,000 (at m/z 200), target automatic gain control (AGC) value of 5e5 and a maximum injection time of 50 ms. Ions selected under top-speed mode were isolated in Quadrupole with an isolation width of 1.6 Da and fragmented by higher energy collision-induced dissociation (HCD) with normalized collision energy of 32%, then measured in the linear ion trap. Typical MS/MS scans mass spectrometric conditions were: targets AGC value of 5e3 and maximum fill time of 35 ms; dynamic exclusion was employed for 18s. The raw data had been deposited to iProX (http://www.iprox.org/index) with the dataset identifier project ID IPX00078900.

Data analysis. The acquired MS/MS spectra were searched by Mascot 2.3 (Matrix Science Inc) implemented on Proteome Discoverer 2.0 (Thermo Fisher Scientific) against the human National Center for Biotechnology Information (NCBI) RefSeq protein databases (updated on 04-07-2013, 32015 protein entries). The parameter settings were: the mass tolerances were 20 ppm for precursor and 0.5 Da for product ions; two missed cleavages were allowed; dynamic modification for TOT were phosphor(Y), phosphor(ST), acetyl(protein N-term), deStreak(C) and oxidation(M); 7


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A false discovery rate (FDR) of 1% was applied at the peptide level. Matching between runs was performed with biological repeats of EGF treatment samples. Protein quantification and absolute amount estimation was described previously11.

Results and discussion A streamlined approach of TFRE on Tip (TOT) Batches of ready-to-use TOT tips were packed and stored at 4°C up to 30 days. Nuclear extracts prepared from cell, organ, and biopsy samples were loaded onto the TOT tips directly. After a series of load-spin-wash steps, DNA-binding proteins captured on beads in TOT tips were subjected to tryptic digestion on tip. Reduction and alkylation reaction steps increased total processing time and reduced the number of TFs identified (Supplementary figure2, Supplementary table 2). Thus, reduction/alkylation reactions were excluded in the TOT approach. The resulting tryptic peptides were either loaded directly to liquid chromatography (LC)-MS for protein identification, or underwent an optional on-tip fractionation by miniaturized basic reverse-phage (RP)-LC before MS analysis to increase the TF coverage (Figure 1). The entire TOT-MS process from sample preparation to data collection can be completed within 3 hours with ~ 1 hour of MS running time. Since all procedures can be executed on the tip, a bench top centrifuge can process 24 samples at a time (Supplementary figure 1), greatly increasing the throughput. If coupled with high throughput robot, the TOT has the potential to handle hundreds of samples 8



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simultaneously.

TOT approach has high reproducibility and sensitivity in TF identification and quantification To investigate the reproducibility of TOT, we performed the entire workflow with 293T and HeLa cell for 3 independent biological replicates. On average, 300 and 176 TFs were identified from 293T and HeLa cells, respectively; in 1-hour MS runs (Figure 2a, Figure 2b, and Supplementary table 1). The Pearson correlation coefficients (R2) between replicates were greater than 0.970 (Figure 2c). We examined 9 well-characterized TFs across four orders of magnitude of abundance and found that quantification was highly reproducible (Figure 2d). We tested the durability of pre-packed TOT tips stored in 4°C refrigerator by repeating the experiments during a 30 day period. After 30 day storage, the number of TFs identified was still comparable to the fresh-made TOT (Figure 2e, and Supplementary table 3).

To evaluate the sensitivity and linearity of TOT, we carried out a series of titration experiments using different amount of NE. As shown in Figure 3a, over 75 and 150 TFs were detected from 500ng and 1µg of total NE, respectively in 1-hour gradient. Using label-free quantification, we found that the TF DNA binding activities exhibited linear response in the range of 1 to 100µg of NE (Figure 3b). To achieve 9


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deeper TF coverage, we coupled TOT with an on-tip basic RPLC fractionation (small size reverse-phase, sRP) to separate tryptic peptides into 6 fractions and subjected them to 6 MS runs with 1 hr gradient each. A total of 757 TFs were identified in 293T cells, encompassing 6 orders of magnitude in abundance (Figure 3c, and Supplementary table 4). When compared to a published mRNA-seq dataset

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that identified 836 TFs (fragments per kb of exon per million mapped fragments, FPKM > 1), the overlap between mRNA-seq and TOT-sRP is 513 TFs (Figure 3d), demonstrating the remarkable sensitivity of TOT in detecting endogenous TFs at protein level. Thus, while the streamlined procedure is of features high efficiency and throughput, the depth of TF profiling by TOT can be further improved with additional steps and more time, to achieve the depth TF identification that is comparable to RNA-seq.

The feasibility of TOT in screening TFs from micro amount of cell and tissue samples We explore the utility of TOT, using commonly available biological materials in basic research and clinical applications. We prepared NEs from cells grown in a single well of 6-, 24-, or 96-well plates and performed TOT-MS and detected 378, 350, and 205 TFs with 1-hour MS run, respectively (Figure 4a, and Supplementary table 5). Alternatively, whole cell extract (WCE) also could be used for TOT, as 216 and 169 TFs were detected from cells grown in a single well of 24- or 96-well plates, 10



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respectively; further streamlining the overall procedure of TOT but with sacrificed sensitivity. We used nuclear extracts from stomach mucosa obtained by gastric endoscope to test clinical sample applications13. In this case, we used a piece of gastric endoscope sample weighing approximately 7-8 mg and detected 140 TFs (Figure 4b, and Supplementary table 6). The whole process, from fresh biopsy sample collection to quantitative TF data production, took as little as 4 hours. These results demonstrated that TOT is a practical approach to detect active endogenous TFs, which can be used not only in laboratory research, but drug screening and clinical diagnosis.

Analysis of temporal changes of global TF-DNA binding patterns after EGF treatment. Because of the simple procedure and short operation time, TOT has high reproducibility and accuracy in TF binding activity quantification. To explore the utility of TOT in determining signal-dependent TF changes, we treated HeLa cells in 6-well plates with EGF for 0, 1 min, 20 min and 120 min, and carried out TOT-MS assays. Three biological repeats of each time point showed high correlation and co-clustering (Supplementary figure 3a and 3b). Among the 281 TFs identified across 4 time points, we classified 10 groups with k-means clustering algorithm, in which classes 1, 3, and 9 showed stimulation in response to EGF treatment. (Figure 11


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4c, Supplementary figure 3c and Supplementary table 7). The GO/pathway analysis indicated that TFs in classes 1, 3, and 9 are significantly enriched in EGF signaling pathway. The dynamic responses of 14 representative TFs showed that the EGF activated TFs, include many well-characterized responders( e.g., FOS14, JUN15, NR4A16, MYC17), and also TFs that were not reported before(e.g., NR2C1, ZBTB2 and HOXC13) (Figure 4d). The potential involvement of the new responders in the EGF signaling pathway warrant further biochemical and functional validations. We summarized the signal transduction pathway of TFs induced by EGF in the Figure 4e. Conclusion In summary, we developed a TOT workflow to facilitate proteome scale TF identification and DNA binding activity quantification. The TOT coupled with MS provides a rapid, sensitive and high throughput method that allows for in-depth interrogation of the TF sub-proteome. These features will render it suitable for dissecting cellular signaling landscape in basic research, drug mechanism screening in industry, and clinical applications, such as tumor molecular typing and diagnosis.

References (1) Naef, F.; Huelsken, J. Nucleic Acids Res. 2005, 33, e111-e111. (2) Zhang, W.; Morris, Q. D.; Chang, R.; Shai, O.; Bakowski, M. A.; Mitsakakis, N.; Mohammad, N.; Robinson, M. D.; Zirngibl, R.; Somogyi, E. J. Biol. 2004, 3, 1 12



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(3) Takahashi, K.; Yamanaka, S. Cell 2006, 126, 663-676 (4) Stormo, G. D.; Zhao, Y. Nat. Rev. Genet. 2010, 11, 751-760 (5) Biggin, M. D. Dev. Cell 2011, 21, 611-626 (6) Vaquerizas, J. M.; Kummerfeld, S. K.; Teichmann, S. A.; Luscombe, N. M. Nat. Rev. Genet. 2009, 10, 252-263. (7) Johnson, D. S.; Mortazavi, A.; Myers, R. M.; Wold, B. Science 2007, 316, 1497-1502. (8) Berger, M. F.; Badis, G.; Gehrke, A. R.; Talukder, S.; Philippakis, A. A.; Pena-Castillo, L.; Alleyne, T. M.; Mnaimneh, S.; Botvinnik, O. B.; Chan, E. T. Cell 2008, 133, 1266-1276. (9) Tan, K.; Feizi, H.; Luo, C.; Fan, S. H.; Ravasi, T.; Ideker, T. G. Proc. Natl. Acad. Sci. USA 2008, 105, 2934-2939 (10) Ding, C.; Chan, D. W.; Liu, W.; Liu, M.; Li, D.; Song, L.; Li, C.; Jin, J.; Malovannaya, A.; Jung, S. Y. Proc. Natl. Acad. Sci. USA 2013, 110, 6771-6776 (11) Ding, C.; Jiang, J.; Wei, J.; Liu, W.; Zhang, W.; Liu, M.; Fu, T.; Lu, T.; Song, L.; Ying, W. Mol. Cell. Proteomics 2013, 12, 2370-2380 (12) Ramos, M.-P.; Wijetunga, N. A.; McLellan, A. S.; Suzuki, M.; Greally, J. M. Epigenet. Chromatin 2015, 8, 1 (13) Debray, C.; Housset, P.; Martin, E.; Bourdais, J. P.; Nicolaidis, C. L. Gut 1962, 3, 273. (14) Peng, C.; Zeng, W.; Su, J.; Kuang, Y.; He, Y.; Zhao, S.; Zhang, J.; Ma, W.; Bode, A. M.; 13


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Dong, Z.; Chen, X. Oncogene 2016, 35, 1170-1179. (15) Bae, J. A.; Yoon, S.; Park, S.-Y.; Lee, J. H.; Hwang, J.-E.; Kim, H.; Seo, Y.-W.; Cha, Y. J.; Hong, S. P.; Kim, H. Clin. Cancer. Res. 2014, 20, 4115-4128 (16) Strom, B. O.; Paulsen, R. E. Biochem Biophys Res Commun 2012, 417, 1292-1297. (17) Li, X.; Liu, X.; Xu, W.; Zhou, P.; Gao, P.; Jiang, S.; Lobie, P. E.; Zhu, T. J. Biol. Chem. 2013, 288, 18121-18133

Acknowledgements This work was supported by Ministry of Science and Technology of China (2016YFA0502500), National Program on Key Basic Research Project (973 Program, 2012CB910300,

2014CBA02000),

International

Collaboration

Grant

(2014DFB30010), National Natural Science Foundation of China (31270822), National institute of health (Illuminating Druggable Genome, U01MH105026) and a grant from the State Key Laboratory of Proteomics (SKLP-YA201401). Author contributions: C.D., J.Q. and F.H. designed research; W.S., K.L., and Yunzhi W. performed research; L.S., M.L. and Z.Q. contributed new reagents/analytic tools; C.D., W.L., X.X., B.Z., F.H. and Yi W. analyzed data; and C.D., Yi W., J.Q. and F.H. wrote the paper. Notes: The authors declare no competing financial interest. 14



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Supporting Information: Supplementary figure1 : TOT approach is amenable to automation and scaling. Supplementary figure2 : TOT approach is high-efficacy and steady. Supplementary figure3 : High reproducibility of dynamic TF patterns following EGF activation indicated by TOT-MS approach. Supplementary table 1 : List of TFs in repeats experiments. Supplementary table 2 : List of TFs in experiments with reduction and alkylation reaction. Supplementary table 3 : List of TFs in storage experiments. Supplementary table 4 : List of TFs in deep coverage experiments. Supplementary table 5 : List of TFs in multi-well plates experiments. Supplementary table 6 : List of TFs in gastric endoscope experiments. Supplementary table 7 : List of TFs in EGF treatment experiments.

Figure legends Figure 1. Streamlined workflow for the TFRE on Tip (TOT) approach. The TOT tips were prepared in advance and stored in the refrigerators before use. Nuclear extract (NE) or whole cell extract (WCE) are directly added into a TOT tip and then are processed described in Experimental section. Samples can be prepared from cell lines, tissues, and biopsies. Basic-RPLC is optional for increasing the coverage of TFs. The whole process can be accomplished within 3 hours. 15


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Figure 2. High reproducibility in the TF identification and quantification by TOT. (A) Numbers of TFs identified in three biological repeats of TOT experiments with 293T or HeLa NE. (B) Venn diagram of identified TFs from three biological TOT repeats with HeLa and 293T NE samples. (C) Correlation of TFs abundances among three biological repeats with 293T NE. R2 values were calculated and indicated. (D) iBAQ values of 6 representative TFs that cross 4 orders of magnitudes in three 293T biological replicates. (E) TFs numbers detected with the TOT tips, which were stored in the refrigerator for 0, 7, 14, 21, and 28 days.

Figure 3. High sensitivity and linearity of the TOT approach. (A) TF numbers detected with different amount NE. Over 200 TFs were identified from 1µg of NE with TOT approach. With sRP pre-fractionation, 750 TFs were identified from 250µg of NE in 6 hours of MS time. (B) Linearity of TOT strategy evaluated by titration analysis. Serial diluted NE were used as shown. iBAQs of representative TFs were calculated. iBAQ value of 1µg NE sample were set as 1 and others were normalized to their corresponding TFs in 1µg NE sample. (C) Dynamic range of TFs identified by TOT-sRP combined approach. Proteins were ranked according to their iBAQ values. (D) Comparison of TF identifications by TOT and RNA-Seq. Venn diagram of the number of expressed TF genes on the protein level and on the mRNA level.

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Figure 4. The wide applications of TOT in dissecting and monitoring the TF patterns of cell line and biopsy. (A) Cells cultured in the 6-, 24-, and 96-well plates were harvested. NE and WCE were prepared from a single well of 6-, 24-, and 96-well plates, and then submitted for TOT. 200-400 TFs were identified from a single well of 6-, 24-, and 96-well plates with 1 hour LC gradient. (B) TF patterns of clinical biopsy samples identified by TOT-MS. More than 130 TFs were detected from 7-8mg of total gastric endoscope weight. (C) Dynamic of TF patterns in HeLa cells treated with EGF. TFs that annotated with EGF signaling pathway were gradually stimulated. (D) Temporal profiles of the representative 14 TFs induced by EGF. (E) Model for temporal regulation of TFs following EGF activation.

For Table of Contents Only

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Analytical Chemistry Figure 1

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A Tissue

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10

0n 20 g 0n 50 g 0n g 1µ g 5µ g 10 µg 50 µ 10 g 0µ 25 2 0 g 0µ 0 µ g g + sR P

0

7

5

25

8

2

50

9

3

200

ADNP ATF1 BZW1 CREB5 ESRRA FOXJ2 GATAD2B MAX NFIA PRDM10 SMAD4 JUN NR2F2 NKX2-5 YY1

10

1

Number of TFs

B

800 700 600 500 400 400

Relative abundance normalized to 1µg NE (Log2)

A 1 2 3 4 5 6 7 8 9 10 11 12 13 14 C 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34


NE amount (log2(µg))

D 1.1 1009 SMARCE1 HMGA1 HMGA2 NR2F2 ZNF644

iBAQ Value

3.4 1007

TOT FDR = 1%

1.0 1006 SIX1

FOXD1 MEF2D TWIST2

HSF2

244

3.3 1004 ZSCAN22 GRHL1 ZBTB16 ZNF410 ZNF599 ZNF701 ZNF155

1.0 1003 3.2 1001

1

RNA-seq FPKM > 1

Rank

755


513

323


A

Page 22 of 21

B

NE TOT NE

En do sc op En e do #1 sc op e #2

Number of TFs

w el lp la 24 te w el lp la te

96

0'

6 107

1'

120'

20'

1.5 106

4 107 2 107

iBAQ Value

1 107

1.0 106

8 106 6 106

5.0 105

4 106 2 106

0.00

3.00

1 TA M

2

13 XC HO

1

3

TB

2C R

ZB

N

AT

2

4A

4A R N

R

N

1

4A

R

N

N

YC

JU

M

B

N

FO SL 1

FO S

FO SB

3.00

F3

0.0

0

JU

Number of TFs

w el lp 24 la te w el lp la 6 te w el lp la te

96

Endoscope Mucosa

Number of TFs

96 well plate 24 well plate 6 well plate 1 2 3 4 NE WCE 5 NE WCE 400 150 250 6 200 7 300 100 8 150 200 9 100 50 10 100 50 11 0 0 12 0 13 14 15 C 16 D 17 EGF 18 19 0 min 1 min 20 min 120 min 20 TOT 21 0’ 1’ 20’ 120’ 22 23 24 25 E 26 27 28 29 30 31 32 ACS Paragon Plus Environment 33 34

Transcription Factor Response Elements on Tip: A Sensitive

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