SUPPLEMENTAL TABLES Supplemental Table 2. Targets of … · 2014. 1. 30. · SUPPLEMENTAL TABLES Supplemental Table 2. Targets of Dnmt3b activity as determined by MSCC analysis. Methylation

SUPPLEMENTAL TABLES

Supplemental Table 2. Targets of Dnmt3b activity as determined by MSCC

analysis. Methylation readout of 260 genes demethylated in MYC;Dnmt3b-/- tumors

relative to MYC;Dnmt3bF/F tumors. The samples are designated as follows: normal

thymocytes (Th), MYC;Dnmt3bF/F tumors (WT), MYC;Dnmt3b-/- tumors (3b), and 24 day

pre-tumor Dnmt3b-deficient thymocytes (3b24).

Supplemental Table 3. Gene expression analysis of genes with significant change

in expression as determined by microarray. Global expression patterns of all

significant changes by microarray were obtained by comparing two normal thymocytes

(N), three MYC;Dnmt3bF/F tumors, and three MYC;Dnmt3b-/- tumors. P-values are

indicated (P<0.05).

Supplemental Table 6. Primer List. List of primers used in this study for COBRA,

genotyping, real time PCR, qRT-PCR and sequencing.

SUPPLEMENTAL MATERIALS AND METHODS

Bisulfite Pyrosequencing of SINE Elements

Pure genomic DNA was treated with sodium bisulfite using the Epitect Bisulfite Kit

(Qiagen). This process deaminates unmethylated cytosine residues to uracil leaving

methylated cytosine residues unchanged. Methylated mouse DNA acquired from NEB

(Ipswich, MA) using M. SssI (CpG) methylase, was treated with sodium bisulfite as

described before served as the positive control. PCR reactions were performed in a

total volume of 25 µl for 35 cycles using Qiagen HotStarTaq DNA Polymerase (1.0U),

MgCl2 solution (3.5 mM), dNTP’s (0.2 mM), sense primer

(TTTTGGTTGTTTTGGAATTTATT, 0.24 uM), antisense primer (5-bio-

CCTTTAATCCCAACACTTAAAAAA, 0.18 µM), with denaturation at 95 ºC for 30

seconds, annealing temperature at 60 ºC for 45 seconds, and extension at 72 ºC for 1

minute. All PCR products were electrophoresed on 0.8% agarose gel, stained with

ethidium bromide, and visualized for appropriate and pure product before proceeding

with all analyses using a Bio-Rad Laboratories Gel-Doc UV illuminator. Methylation

percentage of each CpG was determined using a Qiagen Pyromark Q24 pyrosequencer

and primers (ATTCTGTAGATTAGGTTGGT) according to manufactures

recommendations. The analysed sequence was TTYGAATTTAGAAATTYGTT.

Western Blotting

The following antibodies were used for Western blot: Dnmt3b (52A1018, Imgenex),

Dnmt1 (H-300, Santa Cruz) Dnmt3a (H-295, Santa Cruz), β-Actin (R-22, Santa Cruz)

and c- MYC (9E10, Santa Cruz). For Western blotting, protein lysates were separated in

SDS acrylamide gels and blotted into Immobilon P membranes (Millipore). Blots were

incubated in blocking buffer (5% skim milk) at a concentration of 1 mg/mL. The primary

antibody was then detected using horseradish-peroxidase-conjugated secondary

antibodies and the ECL reagent as described by the manufacturer (Pierce).

Quantitative Real-Time RT-PCR (qRT PCR)

qRT-PCR was performed as described previously (1). cDNA was synthesized from 2 µg

of total RNA using Superscript III reverse transcriptase (Invitrogen) at 50°C for 60 min.

Generated cDNA samples were mixed with gene-specific primers along with SYBR

Green Supermix (Bio-Rad) in a final volume of 20 µL. qRT-PCR was performed in the

CFX96 real time detection system (Bio-Rad). Standard curves were generated using

cDNA to determine the linear range of amplification for each primer pair. Reactions were

performed in duplicate, and relative amounts of cDNAs were normalized to the

expression of Gapdh, or β-Actin.

siRNA and shRNA knockdown experiments

SMARTpool siRNA designed against mouse Gm128 (Ment), Rpl39l, Psma8, Pnldc1 and

Syce1 along with non-silencing RNA, siGLO green transfection indicator and the

DharmaFECT 4 reagent were purchased from Dharmacon. SiRNA at a final

concentration of 100 nM was transfected into 5 x104 MYC;Dnmt3b-/- cells along with

siGLO green to monitor transfection efficiency that was found to be typically ~80%.

Cells were counted 48 h later using a hemacytometer. Non-silencing RNA served as a

negative control. Each experiment was done in duplicates and repeated three times.

Knockdown efficiency was confirmed by qRT-PCR. The same approach was used to

transfect SMARTpool siRNA against CIorf56 (MENT) into JURKAT cells. HuSH 29-mer

shRNA constructs against mouse Gm128 (Ment) were purchased from Origene.

Production of retroviruses was achieved in Phoenix-Eco packaging cell line. The virus

was then used to infect MYC;Dnmt3b-/- cells as described above. Cells were placed on

puromycine selection 36 h after infection for two days. Freshly selected cells were

plated at concentration of 0.2 x 106/mL and counted using hemacytometer. Each

counting was done in triplicates every 12 h to generate growth curves.

Combined Bisulfite Restriction Analysis (COBRA) and Bisulfite Sequencing

Bisulfite conversion of genomic DNA was carried about by sodium bisulfite treatment

using the Epitect Bisulfite Kit (Qiagen). Bisulfite primers were designed using

MethPrimer (2). A fully methylated CpG control was purchased from NEB. PCR

products were digested with restriction enzymes BstUI, TaqI (both from NEB), and Tail

(Fermentas). Digested products were then loaded on an 8% PAGE gel, separated by

electrophoresis and stained by SYBR Green Gold (Invitrogen). PCR fragments from

bisulfite treated DNA were cloned using pGem Easy T-cloning kit (Promega). Plasmid

DNA isolated from individual clones was sequenced at the UNMC Sequencing Core

Facility.

Array Comparative Genomic Hybridization (aCGH)

aCGH was performed at the Comparative Molecular Cytogenetic Core at NIH, NCI at

Frederick using 4x44K arrays from NimbleGen on DNA samples isolated from three

MYC;Dnmt3bF/F and three MYC;Dnmt3b-/- lymphomas. DNA isolated from the kidney of

EμSR-tTA;Teto-Cre;Rosa26LOXPEGFP mouse served as a control.

SUPPLEMENTAL REFERENCES

1. Opavsky R, Tsai SY, Guimond M, Arora A, Opavska J, Becknell B, Kaufmann M, Walton NA, Stephens JA, Fernandez SA, et al. Specific tumor suppressor function for E2F2 in Myc-induced T cell lymphomagenesis. Proc Natl Acad Sc. 2007;104(39):15400-15405.

2. Li LC, Dahiya R. MethPrimer: designing primers for methylation PCRs. Bioinformatics. 2002;18(11):1427-1431.

3. Rai K, Sarkar S, Broadbent TJ, Voas M, Grossmann KF, Nadauld LD, Dehghanizadeh S, Hagos FT, Li Y, Toth RK, et al. DNA demethylase activity maintains intestinal cells in an undifferentiated state following loss of APC. Cell. 2010;142(6):930-942.

Hlady_Supplemental_Table_1

Supplemental Table 1. Table of cytogenetic aberrations identified using array Comparative Genomic Hybridization. Aberrations for three MYC;Dnmt3bF/F (1‐3) and three MYC;Dnmt3b‐/‐ (4‐6) tumors. Positions of amplifications and deletions are given in both chromosomal location and cytoband. The number of probes corresponding to each amplification or deletion is shown along with a P‐value.

(#1)Event Chr Cytoband #Probes Amp/Del P-value Annotations

1 chr6:40826823-40978497 qB1 7 -0.69984 1.72E-11 599, 1700074P13Rik, 1810009J02 chr6:41184457-41451795 qB1 6 -2.25136 7.94E-49 Try4, 1810049H19Rik, Try10... 3 chr13:19196860-19197260 qA2 1 -3.61305 4.27E-144 chr14:52754373-53101965 qC2 5 -2.59613 4.75E-365 chr17:3075528-93509199 qA1 -qE5 1752 0.334409 0.00E+00 Tiam2, Tiam2, Tfb1m...

(#2)Event No Chr Cytoband #Probes Amp/Del P-value Annotations

1 chr6:41184457-41451795 qB1 6 -0.91349 2.65E-14 Try4, 1810049H19Rik, Try10... 2 chr8:108832681-108833081 qD3 1 -2.26894 4.01E-12 Slc12a4 3 chr11:114487382-121517858 qE2 329 0.414111 5.71E-218 Rpl38, Rpl38, Ttyh2... 4 chr13:19196860-19197260 qA2 1 -3.53667 2.11E-105 chr14:52754373-53101965 qC2 5 -2.88431 5.05E-286 chr17:39671705-81008799 qB1 -qE3 691 0.42206 0.00E+00 Pgk2, Crisp3, Crisp1... 7 chr17:81137009-91151345 qE3 -qE5 133 -0.64555 1.55E-141 Slc8a1, AW548124, Eml4...


1 chr14:53002908-53101965 qC2 3 -3.04096 1.37E-15


1 chr3:142814058-142814458 qH1 1 2.446012 4.10E-11 Pkn2 2 chr5:5754545-5773942 qA1 2 2.144442 2.88E-18 Steap1 3 chr5:22639484-22639884 qA3 1 4.28425 1.06E-15 Lhfpl3 4 chr6:41184457-41451795 qB1 6 -2.13734 1.96E-48 Try4, 1810049H19Rik, Try10... 5 chr6:53994195-54192108 qB3 2 2.927514 1.23E-186 chr6:60656216-60918121 qB3 8 -0.99476 1.87E-22 Snca, Snca 7 chr10:14993972-14994372 qA2 1 2.180098 2.72E-128 chr10:89618998-89619398 qC2 1 2.072192 2.43E-109 chr11:116876097-116876497 qE2 1 3.015854 3.47E-1010 chr11:120426769-120427169 qE2 1 -2.46261 2.79E-16 Pcyt2 11 chr14:53065963-53101965 qC2 2 -3.21457 1.36E-1812 chr15:3228882-103359097 qA1 -qF3 1663 0.452136 0.00E+00 Sepp1, Sepp1, Sepp1...


1 chr3:127753730-127754130 qG2 1 -4.14299 1.96E-132 chr3:142814058-142814458 qH1 1 2.788929 1.06E-15 Pkn2 3 chr5:22639484-22639884 qA3 1 3.319846 2.20E-10 Lhfpl3 4 chr6:41184457-41451795 qB1 6 -3.36505 4.85E-71 Try4, 1810049H19Rik, Try10... 5 chr6:53319496-53319896 qB3 1 -2.23691 7.14E-116 chr6:54191708-54192108 qB3 1 5.100765 1.30E-197 chr10:89618998-89619398 qC2 1 1.832661 2.72E-108 chr14:52754373-53101965 qC2 5 -3.9517 3.08E-499 chr15:54493923-54494323 qD1 1 2.839228 1.60E-1110 chr19:10273547-10273947 qA 1 2.189765 3.42E-1211 chr19:13937681-13938081 qA 1 2.913638 1.12E-1412 chrX:68840554-76033941 qA7.3 -qB 132 0.789382 3.46E-276 Gabra3, Gabrq, Magea9...


1 chr3:45382016-45626918 qB 5 0.68297 1.00E-11 Pcdh10, Pcdh10, Pcdh10... 2 chr4:136972899-136973299 qD3 1 2.023405 7.43E-12 Rap1gap 3 chr5:5754545-5773942 qA1 2 2.382502 8.41E-21 Steap1 4 chr5:22639484-22639884 qA3 1 4.330955 4.57E-17 Lhfpl3 5 chr6:41184457-41451795 qB1 6 -3.16525 2.49E-68 Try4, 1810049H19Rik, Try10... 6 chr6:53319496-53319896 qB3 1 -2.41258 3.25E-117 chr6:54191708-54192108 qB3 1 4.654747 6.80E-198 chr6:121072205-121072605 qF1 1 -3.85925 2.12E-109 chr10:14993972-14994372 qA2 1 3.046566 2.40E-1510 chr11:115235363-115235763 qE2 1 2.208779 7.62E-12 Atp5h 11 chr11:116876097-116876497 qE2 1 3.652867 1.57E-1212 chr13:19196860-19197260 qA2 1 -4.69105 2.34E-1613 chr14:52754373-53101965 qC2 5 -3.89983 5.87E-5014 chr19:5706829-5707229 qA 1 3.488686 7.69E-17 Kcnk7 15 chr19:8588764-8589164 qA 1 3.413205 7.78E-1116 chr19:10273547-10273947 qA 1 2.422973 5.19E-1317 chr19:11530152-11530552 qA 1 4.064957 2.97E-14 Ms4a4b 18 chr19:13477369-13477769 qA 1 2.285003 3.43E-11 Olfr1469 19 chr19:13937681-13938081 qA 1 3.318061 4.70E-16


-12 +12

Supplemental Table 4. Significantly deregulated genes in MYC;Dnmt3b‐/‐ tumors as determined by microarray: A heatmap displaying 40 genes that are 3‐fold or more up‐regulated in MYC;Dnmt3b‐/‐ tumors relative to MYC;Dnmt3bF/F tumors as well as 25 genes showing a 3‐fold or greater decrease in expression (P<0.01). A color bar is shown to reference fold changes with red being up‐regulated, and green being down‐regulated. An average of two normal thymocytes (N) serve as a control for pairwise comparison of three MYC;Dnmt3bF/F and three MYC;Dnmt3b‐/‐ tumors.

Gene ThRpl39l 1 ‐1.15562 ‐1.0253 ‐1.06905 7.446746 39.98144 32.09777 <0.001Pnldc1 1 1.047398 1.065331 1.297218 28.04891 32.85522 32.55415 <0.001Syce1 1 ‐1.07755 1.01264 1.017302 36.96391 23.22533 12.18671 <0.001Gm4638 1 1.175584 1.703288 1.204562 5.721351 41.34575 48.20938 <0.001S100a6 1 ‐1.09384 ‐1.23316 1.08505 7.742137 18.95699 7.942287 <0.001Tns4 1 1.522487 1.648387 1.838878 37.4808 10.6516 6.672788 <0.001Epha3 1 1.014144 1.01945 1.002131 14.78668 10.72752 4.944101 <0.001Atp1a3 1 ‐1.0253 1.571848 ‐1.10419 20.37385 6.044364 3.395995 <0.001Rbpms 1 1.411509 1.795107 1.046587 16.20619 12.80345 5.128716 <0.001Apobec2 1 ‐1.08426 1.127483 1.014133 14.40467 3.144442 4.92049 <0.001Ass1 1 ‐1.46953 1.375223 ‐2.60408 5.085414 6.943577 4.219163 <0.001Qser1 1 ‐1.19144 ‐1.67619 ‐2.34626 4.758856 2.523249 4.433712 <0.001Myadm 1 1.51187 ‐1.3466 1.439309 14.92979 5.234661 3.05054 <0.001Mt2 1 4.987487 1.920478 4.876631 56.5647 8.190547 4.812219 0.003Brdt 1 ‐1.29355 ‐1.08964 ‐1.07868 3.330702 4.349762 7.080151 <0.001

700029I01R 1 1.471148 1.223514 1.739871 10.64083 6.799094 5.20817 <0.001Pkn3 1 ‐1.17639 ‐1.24369 ‐1.22728 2.7475 3.467984 5.516675 <0.001

Gm13051 1 2.050126 1.281614 2.965273 15.40008 6.903724 7.514142 <0.001Gstm1 1 2.502135 ‐1.42614 1.449584 9.139796 9.024985 3.612881 <0.001Enpp1 1 1.702507 7.879489 2.310974 26.05383 14.14353 14.1068 <0.001

Gm13251 1 2.358147 ‐1.77805 2.475884 11.44664 5.960746 6.03484 <0.001Tex14 1 1.105546 1.042973 2.112595 1.525261 8.490341 6.571019 0.002Spry2 1 ‐1.40773 ‐1.44042 ‐1.20463 3.646293 3.353952 1.537365 <0.001Psma8 1 1.065959 1.077746 1.069802 1.153524 3.507901 7.516588 0.0020Chchd10 1 1.485018 1.540731 1.33092 11.22342 1.649978 2.840991 <0.01Myo1e 1 2.236682 3.175779 1.130977 7.28581 9.136728 7.169814 <0.001Ms4a4b 1 2.026883 ‐1.59139 2.866461 11.35861 5.778969 2.612381 0.001BC021614 1 ‐1.35158 3.557845 1.646253 14.73779 3.630382 2.872842 <0.01Cd44 1 1.279539 1.027857 2.430472 11.29092 2.139615 2.87386 <0.01

Snord82 1 ‐1.06156 1.208677 ‐1.21639 6.80872 1.834695 1.567541 <0.01LOC629446 1 1.466454 ‐2.36424 ‐1.1192 2.746599 4.608141 1.885147 <0.001

Bag3 1 1.788389 1.686495 1.866465 11.18465 3.653912 2.850029 <0.01Adi1 1 1.646629 1.58198 1.304855 10.26548 2.096143 2.607854 <0.01Klf9 1 1.596809 2.678958 1.615968 9.534676 5.571789 4.272453 <0.001

Slc22a15 1 ‐1.29721 1.123893 ‐1.139 3.324351 3.076536 2.498632 <0.001Hdc 1 8.961861 14.30646 ‐1.0547 23.50522 28.44498 25.11063 <0.001

Gm128 1 ‐1.24906 ‐1.12765 ‐1.12222 2.656525 3.475067 1.924004 <0.001Slc39a4 1 1.143677 ‐1.15965 1.337107 5.555395 2.801118 1.971126 <0.001Prr5 1 2.542029 1.147679 2.505571 13.2486 2.94192 2.868272 0.01Cebpa 1 1.845383 2.043873 1.066898 4.885597 4.91725 5.10445 <0.001Gng3 1 5.016985 1.583372 6.69806 1.23458 1.31599 1.662918 <0.001Pja2 1 4.99261 4.828205 13.55035 2.005918 1.878191 3.2741 <0.001

Serpine2 1 3.340198 6.980948 3.646376 ‐1.02829 1.583021 1.67583 <0.001Crtam 1 9.789362 11.20017 15.78151 1.176679 5.398249 4.424684 <0.001Lmo2 1 13.70378 3.339565 12.79892 1.749874 4.14701 2.813448 <0.001Eomes 1 4.791118 2.012904 6.682684 ‐1.14773 1.125798 ‐1.09074 <0.001

430407P10R 1 18.93511 3.06024 23.94598 ‐1.08239 5.29791 3.638173 <0.01Spock2 1 14.52922 3.360568 23.90337 1.973858 5.096115 1.679154 <0.001Rhob 1 14.94211 1.954319 19.02698 1.284056 4.722185 1.488758 <0.01Fbn2 1 2.981732 7.154415 33.37297 1.021369 5.393258 2.020782 <0.01Ptpn14 1 3.344837 2.098433 5.849958 ‐1.99939 ‐1.54206 1.008725 <0.001Rnf144b 1 17.8035 2.967845 14.1918 4.380415 ‐1.0305 ‐1.02363 <0.001Chst2 1 9.930992 4.888061 19.17715 1.186588 3.285927 1.622769 <0.001Gpr87 1 13.68737 2.017384 1.768283 ‐1.13284 ‐1.04068 1.054432 <0.001Grb10 1 163.6838 35.88767 213.5595 ‐1.23506 27.50859 40.20214 <0.001

Sh3bgrl2 1 16.79583 1.535852 18.72166 ‐1.01153 2.188925 2.471064 <0.01Upb1 1 13.14367 2.579203 5.151394 ‐1.2251 ‐1.06658 ‐1.06123 <0.001Etv5 1 19.97266 4.513352 21.84494 1.029946 1.783429 2.794386 <0.001Palmd 1 24.75831 1.744318 27.33398 1.224537 2.960916 1.765643 <0.001Cacna1b 1 6.22978 2.639605 35.70118 1.695525 1.417433 1.73528 <0.001Efhd1 1 18.41263 1.522964 40.70466 1.102636 4.19791 1.17829 <0.01Tnni1 1 21.5886 1.955816 42.3564 1.058616 3.57441 1.361819 <0.001Slc7a8 1 10.50429 1.597538 9.09238 ‐1.76299 ‐1.48413 ‐1.70048 <0.001Hspa1a 1 22.15759 19.72648 5.274207 1.018381 1.194695 1.227554 <0.001Wif1 1 101.9889 15.90264 112.6128 ‐1.02577 2.301956 3.115846 <0.001

MYC;Dnmt3bF/F MYC;Dnmt3b‐/‐


Supplemental Table 5. Overlapping targets of Dnmt3b and “active” demethylase

activity. Out of 5315 genes found to be targets of active DNA demethylases (3), 18

genes were found to be targets of Dnmt3b methylation activity in this study.

Epo Arhgef18 Fpgs Mobkl2c Otof Tekt2

Nat11 Cpt1b Guca1b Mtmr9 Piwil1 Trpv6

Acvrl1 Ddost Mcm7 Numbl Psma8 Utp11l

Thymus Spleen Lymph Node % % %

EGFP+ DN CD4 CD8 CD4/CD8 EGFP+ DN CD4 CD8 CD4/CD8 EGFP+ DN CD4 CD8 CD4/CD8

Mutant 9+/-8 22+/-11 3+/-2 66+/-16 Mutant 78+/-5 17+/-5 5+/-1 0+/-0 Mutant 27+/-4 53+/-6 19+/-2 2+/-0

Control 7+/-2 18+/-4 3+/-2 72+/-0 Control 71+/-6 21+/-5 8+/-2 0+/-0 Control 25+/-8 54+/-8 18+/-1 3+/-1

% % %

EGFP- DN CD4 CD8 CD4/CD8 EGFP- DN CD4 CD8 CD4/CD8 EGFP- DN CD4 CD8 CD4/CD8



% % %

EGFP+ DN CD4 CD8 CD4/CD8 EGFP+ DN CD4 CD8 CD4/CD8 EGFP+ DN CD4 CD8 CD4/CD8



% % %

EGFP- DN CD4 CD8 CD4/CD8 EGFP- DN CD4 CD8 CD4/CD8 EGFP- DN CD4 CD8 CD4/CD8



STOP

EμSRα tTa

Teto Cre

ROSA EGFP

Dnmt3b

A B C

KO

F

0

120

90

60

30

Dnmt3b-/- F/+ F/F

% o

f C

onditio

nal A

llele Dnmt3b-/-

EµSR-tTA;Teto-Cre;Rosa26LOXPEGFP;Dnmt3bF/F

EGFP+, Dnmt3b-deficient EGFP-

tTA+ tTA-

EGFP+ EGFP-

EµSR-tTA;Teto-Cre;Rosa26LOXPEGFP;Dnmt3b+/+

(Control) (Mutant)

CD

4

CD8

tTA+ tTA-

Hlady_Supplementary_Fig_1

Thymus Spleen Lymph Node

Time (months)

% S

urv

ival

0 2 4 6 8 10 12

100

75

50

25

0

Dnmt3b-/- (n=12)

Dnmt3b+/+ (n=12)

D

E

F

G

Genotype Number %EGFP+

Thymus Cells

(x10^6)

Thymus

Weight

Spleen Cells

(x10^6) Spleen Weight

Lymph Node

Cells (x10^6)

Lymph Node

Weight

Mutant 9 49+/-10 301+/-97 0.1+/-0.02 299+/-44 0.1+/-0.01 45+/-12 0.03+/-0.01

Control 6 42+/-10 332+/-162 0.1+/-0.02 348+/-54 0.15+/-0.05 45+/-9 0.04+/-0.01

Mutant 5 55+/-11 318+/-351 0.05+/-0.02 167+/-156 0.15+/-0.01 105+/-88 0.02+/-0.02

Control 4 51+/-9 476+/-288 0.04+/-0.01 226+/-126 0.12+/-0.02 272+/-218 0.03+/-0.02

Early

Late

Ea

rly

La

te

H


Supplemental Fig. 1. Loss of Dnmt3b has no affect on T cell

development. (A) Graphical presentation of EμSRα-tTA, Teto-Cre, and

Rosa26LOXPEGFP transgenes and the Dnmt3b conditional knock out allele

used in developmental studies. The blue color indicates that tTA regulates

transcription from Teto promoters and recombination of lox P sites (triangles).

(B) Deletion efficiency of the conditional allele of Dnmt3b as determined by

real time quantitative RT-PCR (qRT-PCR) analysis on genomic DNA isolated

from EμSRα-tTA;Teto-Cre;Rosa26LOXPEGFP;Dnmt3bF/F (Dnmt3b-/-), EμSRα-

tTA;Teto-Cre;Rosa26LOXPEGFP;Dnmt3bF/+ (F/+) and EμSRα-

tTA;Rosa26LOXPEGFP;Dnmt3bF/F (F/F) mice (n=2 for each). Error bars

represent standard error of the mean (+/- SEM). (C) PCR based analysis of

deletion efficiency of the conditional allele of Dnmt3b (F) in DNA from FACS-

sorted EGFP+ from EμSRα-tTA;Teto-Cre;Rosa26LOXPEGFP;Dnmt3bF/F

thymocytes. (D) Kaplan-Meier survival curves of EμSRα-tTA;Teto-

Cre;Rosa26LOXPEGFP;Dnmt3bF/F (Dnmt3b-/-, red line) and EμSRα-tTA;Teto-

Cre;Rosa26LOXPEGFP;Dnmt3b+/+ (Dnmt3b+/+, blue line) mice. Number of mice

(n) is indicated. (E) Percentage of EGFP+ cells in thymus as well as organ

weights and cellularity for thymus, spleen and lymph nodes isolated from

EμSRα-tTA;Teto-Cre;Rosa26LOXPEGFP;Dnmt3bF/F (Dnmt3b-/-) and EμSRα-

tTA;Teto-Cre;Rosa26LOXPEGFP;Dnmt3b+/+ (Dnmt3bF/F) mice at age 31 days

(early) and one year (late). (F) Schematic representation of inter- and intra-

mouse comparisons. EGFP-positive (EGFP+) and EGFP negative (EGFP-)

populations within an individual mouse are shown by purple (control) and red

(mutant) arrows. In mutant mice, EGFP+ cells will be Dnmt3b-deficient, while

EGFP- cells retain Dnmt3b. Comparisons between control and mutant mice

are denoted by green (EGFP+ to EGFP+) and blue (EGFP- to EGFP-) arrows.

(G) FACS profiles of CD4 and CD8 expression in thymus, spleen, and lymph

node of control and mutant mice. Green color indicates EGFP positive cells,

blue color indicates EGFP negative cells. (H) CD4/CD8 profiles for thymi,

spleens, and lymph nodes for early and late EμSRα-tTA;Teto-

Cre;Rosa26LOXPEGFP;Dnmt3bF/F (mutant) and EμSRα-tTA;Teto-

Cre;Rosa26LOXPEGFP;Dnmt3b+/+ (control) developmental mice as determined

by FACS. Average percentages of double negative CD4-CD8- (DN), single

positive CD4+ (CD4), single positive CD8+ (CD8) and CD4+CD8+ double

positive (CD4/CD8) are shown with standard deviations.

CD4hiCD8+CD4+

2

0

8

6

4

Num

ber

of

Lym

phom

as

Dnmt3bF/F

Dnmt3b-/-

Number of mice CD4hiCD8+ CD4+ CD44hiCD25- CD44loCD25- B220-CD11b-

MYC;Dnmt3b-/- 10 8 2 5 5 10

MYC;Dnmt3bF/F 10 8 2 6 4 10

A few tumors showed a small percentage of B220- and/or CD11b- positive cells

A

B


MYC

Supplemental Fig. 2. Loss of Dnmt3b does not alter the

immunophenotypes of MYC-induced T cell lymphomas. (A)

MYC;Dnmt3bF/F (n=10) and MYC;Dnmt3b-/- (n=10) tumors were stained with

anti-CD4 and anti-CD8 antibodies and categorized as either CD4-single

positive, or CD4hiCD8+ based on FACS. (B) Summary of immunophenotypes

as determined by FACS analysis using CD4, CD8, CD44, CD25, B220, and

CD11b expression in MYC;Dnmt3bF/F and MYC;Dnmt3b-/- tumors.

A

10

/0

9/1

8/2

7/3

6/4

5/5

4/6

3/7

2/8

1/9

0/1

0

Ratio of KO/Conditional Dnmt3b DNA

MYC;Dnmt3b-/-

KO (-)

Conditional (F)


% C

on

ditio

na

l Alle

le

B

F/F F/+ -/-0

20

40

60

80

100

120

Supplemental Fig. 3. Deletion efficiency of Dnmt3b conditional allele in

lymphomas. (A) PCR based analysis of deletion efficiency of the conditional

allele of Dnmt3b in lymphomas (n=5) derived from MYC;Dnmt3b-/- mice

(Dnmt3b-/-). Genomic DNA with differing ratios of Dnmt3b- (ko; -) and Dnmt3bF

(conditional; F) were used as reference samples. (B) Relative levels of the

conditional allele of Dnmt3b as determined by Real time PCR analysis of

genomic DNA in three MYC;Dnmt3bF/F (F/F) , two MYC;Dnmt3bF/+ (F/+) and

three MYC;Dnmt3b-/- (-/-) mice. Averages +/- SEM is shown.

P=0.32

Dnmt3b-/-0

1

2

3

Rela

tive E

xpre

ssio

n

Dnmt3bF/F


MYC

Supplemental Fig. 4. Loss of Dnmt3b does not affect expression of MYC

transgene. Relative expression of the MYC transgene as determined by qRT-

PCR analysis in five MYC;Dnmt3bF/F and MYC;Dnmt3b-/- lymphomas with the

averages +/- SEM shown. No statistically significant difference is seen

(P=0.32).

20

0

40

60

80

CD4+ CD4+CD8+ DN CD8+

% o

f C

ells

Dnmt3b-/-

Dnmt3bF/F


Supplemental Fig. 5. Expression of transgenic MYC does not alter T cell

development. Expression of cell surface markers CD4 and CD8 in 24 day old

MYC;Dnmt3bF/F and MYC;Dnmt3b-/- thymocytes during early tumorigenesis.

Average percentages of double negative CD4-CD8- (DN), single positive CD4+,

single positive CD8+ and CD4+CD8+ double positive are shown with standard

deviations. Error bars represent +/- SEM and no statistically significant

difference is seen.

Spleen

Lymph Node

Time (days)

60

40

20

035 50 Final

% P

rolife

rating

C

ells

Time (days)

60

40

20

035 50 Final

% P

rolife

rating

C

ells

Time (days)

0

5

20

25

15

10

% A

po

pto

tic C

ells

35 50 Final

Time (days)

0

4

8

12

16

% A

po

pto

tic C

ells

35 50 Final

A

B

Dnmt3b-/-

Dnmt3b+/+

Dnmt3b-/-

Dnmt3b+/+


MYC

MYC

*

*

n=

11

n=

9

n=

5

n=

6

n=

6

n=

7

n=

11

n=

9

n=

5

n=

6

n=

6

n=

7

n=

11

n=

9

n=

5

n=

6

n=

6

n=

7

n=

11

n=

9

n=

5

n=

6

n=

6

n=

7

Supplemental Fig. 6. Loss of Dnmt3b results in enhanced proliferation of

tumors in vivo. Apoptosis and BrdU incorporation assay as determined by

FACS analysis of cells isolated form spleens (A) and lymph nodes (B) of

MYC;Dnmt3b+/+ (blue bars) and MYC;Dnmt3b-/- (red bars) mice at age 35 and

50 days as well as terminally ill mice (Final). Anti-Annexin V and anti-BrdU

antibodies were used to evaluate apoptosis and BrdU incorporation,

respectively. The number of mice used for each cohort is indicated by n. Error

bars represent the SEM and significant changes are denoted by (*) P<0.05.

Assay Sine B1

Sample ID: 3b Tumor

Sequence to Analyze: TTYGAATTTAGAAATTYGTT

Assay Sine B1

Sample ID: WT Tumor

Sequence to Analyze: TTYGAATTTAGAAATTYGTT


49% 45%

54% 49%

Supplemental Fig. 7. Methylation analysis of SINE-B1 repetitive elements

by pyrosequencing. A representative pyrogram from one MYC;Dnmt3b-/-

tumor (3b Tumor) and one MYC;Dnmt3bF/F tumor (WT Tumor) from SINE-B1

repeat elements is shown. Percentages represent the amount of

methylcytosine at the highlighted locus.

0

4

567

3

2

1

Arfip 1

0

1

2

3

4

0

2

4

6

8 Fbln2

0

2

4

6

8Mb Mtap7d1

0

1

2

3

4

5

0

3

6

9

12Stx 1

Cutl 1

0

1

2

3

4

Rbbp9

0

20

40

60

80


Fold

induction

0

5

10

15

20

25 Ahi1

A

Angpt4

0

4

8

12

16Rxfp3

Fold

induction

0

0.4

0.8

1.2

1.6

2.0 Phlpp Sla2

0

2

3

1

4

Fold

induction

Commd7

0

1.2

1.8

0.6

Fold

induction

CCGG

CCGG

HpaII digest

Methylated

No digest by HpaII

PCR successful

Not methylated

Digested by HpaII

PCR fails

C CGG

B

C

Gene Sequence Chr Position Strand N1 N2 N3 wt1 wt2 wt3 wt4 wt5 3b1 3b2 3b3 3b4 3b5

Ahi1 AGCCTGCAGAGAGGGAGG 10 20785621 R 20 50 34 2 1 6 32 4 21 6 7 7 14

Angpt4 GTTGGCTTCTCGGTTTGC 2 151765097 R 17 8 13 17 9 11 20 10 1 1 7 7 8

Arfip1 CAGGATCTCTGCAGGTCA 3 85209887 R 12 7 11 3 2 2 13 13 13 14 25 21 23

Commd7 TGGAAAGGGGTGTGGTCA 2 153458100 R 35 7 22 16 16 4 4 1 2 1 2 1 1

Cutl1 ATGCGAGCTGCTCAGGCC 5 136879583 R 3 1 2 1 1 1 3 1 1 1 2 1 1

Fbln2 CCCAGCTGCCCCCCATCC 6 91163042 R 20 31 32 20 17 16 5 1 1 1 1 1 1

Mb GGTGTGGGGCAGCCTGAG 15 76865085 R 2 4 3 8 13 17 3 38 36 26 38 50 3

Mtap7d1 TGTAGAGGGAGAGGATGG 4 125912322 R 17 1 7 4 1 1 3 1 6 10 3 2 3

Phlpp GCTCCATGGAGAGACTAC 1 108069889 R 12 8 10 8 14 2 7 4 1 1 7 6 7

Rbbp9 GAGTTGATGACCCAGAAG 2 144382198 R 42 53 44 18 38 32 27 3 1 1 1 1 1

Rxfp3 CCGCTGTAGACCACCACA 15 10965680 R 21 36 25 18 23 1 34 10 1 4 10 14 8

Sla2 GGCTTTTCTAGCTCACAG 2 156703666 R 72 232 157 57 67 185 46 138 1 1 1 1 1

Stx1 CGCCGAGTGCGCGCCGTG 10 12661433 R 12 1 7 2 2 1 3 4 11 1 2 1 11


Supplemental Fig. 8. Validation of MSCC data by HpaII–based real time

PCR analysis. (A) A schematic for Real-Time PCR confirmation of MSCC

data. HpaII sites that corresponded with significant MSCC data had primers

designed on either side. Genomic DNA was digested with HpaII and qRT-PCR

was performed. Higher levels of PCR product correlated with more highly

methylated samples, as only methylated HpaII sites will yield successful PCR

products. (B) MSCC counts for HpaII sites tested for Real time confirmation.

HpaII sites that did not correlate with MSCC data are marked in red. (C) Real

time PCR analysis of 13 HpaII sites in normal thymocytes (N), MYC;Dnmt3bF/F

(Dnmt3bF/F), MYC;Dnmt3b-/- (Dnmt3b-/-) and fully methylated CpG control

(CpG). An MspI digest, which digests CCGG regardless of its methylation

status, is shown as a negative control. Genes that do not confirm MSCC data

are shown in red. The average values for two replicates are shown with +/-

SEM.

Speg

Cld

n4*

TE

AD

2

N Dnmt3bF/F

CpG

Dnmt3b-/-

BC

035954

Hoxa3

Rspry

1C

lic4

Tra

f3Is

ynA

1Ig

f2bp3

A

B

U

D

U

D

U

D

U

D

U

D

U

D

U

D

U

D

U

D

U

D


MYC

Slc

39a4

Epha3

Pkn3

Ass1

Brd

t*

U

D

U

U

U

U

D

D

D

D

Arh

gef1

8

Vasn*

Pfn

3S

ycp1

†

U

D

U

D

U

D

Pnld

c1

U

D

U

D

U

D

S100a6*

N Dnmt3bF/F

CpG

Dnmt3b-/-

MYC

N Dnmt3bF/F

CpG

Dnmt3b-/-

MYC

N Dnmt3bF/F

CpG

Dnmt3b-/-

MYC

Gene Sequence Chr Position Strand Th1 Th2 Th3 wt1 wt2 wt3 wt4 wt5 3b1 3b2 3b3 3b4 3b5

Arhgef18 TCCAAAGAGTCCGCGACC chr8 3393139 F 3 1 2 4 1 2 3 43 27 33 54 54 41

Ass1 TGGCCATTCTGCCCAAAC chr2 31320382 R 4 3 3 11 2 1 8 4 9 8 26 16 18

BC035954 CGTCCTCGGCCTCCTCCT chr4 148326478 F 3 2 2 2 1 1 3 2 11 11 6 3 1

Brdt GCTGCTGACAGCCGCCAT chr5 107760221 R 1 1 1 1 1 1 1 1 1 13 23 39 1

Cldn4 CCCATTTCCCTCTTCTGC chr5 135422153 R 1 3 1 1 1 1 1 5 7 11 5 7 13

Clic4 CCCAGCGGTTTGAAGCGG chr4 134828698 F 14 53 44 21 29 35 41 15 32 34 43 25 42

Epha3 GCTGGGATAAGGCTTACA chr16 63729522 R 6 5 4 9 1 1 18 7 4 6 26 2 1

Hoxa3 CCGAGTGCCAAGACCAGG chr6 52127507 R 1 1 1 1 1 1 2 32 25 28 34 31 52

Igf2bp3 CGTCTCCGCAGCTGCCAT chr6 49164244 F 12 32 23 17 16 13 16 11 19 21 32 17 9

IsynA1 GGGTCTGGATGAGAACGG chr8 73119104 R 2 38 14 19 16 42 4 9 24 17 55 3 9

Pfn3 CGTCTGCAGAAAGGTGTG chr13 55516361 R 7 2 1 1 3 2 2 18 17 19 35 11 5

Pkn3 CCTAGGTCTGAGAAGCAA chr2 29934592 F 4 3 5 1 1 1 13 9 5 26 59 22 17

Pnldc1 CGCTAGTCCCAGGGCGCG chr17 13102800 R 1 1 1 1 1 1 1 1 1 29 39 27 1

Rspry1 GACTCCCGCGGCTCCGCT chr8 97125226 R 21 11 16 9 11 8 6 3 16 9 8 1 1

S100a6 TCCCTCACCTAAGTTGCC chr3 90417563 R 9 14 11 1 2 5 3 7 9 25 48 5 2

Slc39a4 CCCAGCTCCAGTCTGGCC chr15 76447267 F 13 9 12 23 18 12 1 11 19 27 53 15 1

Speg GGGGGTTCCCGCTTGCGA chr1 75385447 F 8 13 11 1 1 3 3 1 16 11 9 1 1

Sycp1 GGGCCCGTGGTCGCGTGG chr3 102739739 F 1 1 1 1 1 1 1 1 11 4 13 1 1

Tead2 GGGGAGCGCGCGGGGTGG chr7 52471439 R 11 14 9 6 2 4 13 5 13 9 18 5 9

Traf3 CGTCAGCATCTCCGAGGC chr12 112405082 R 37 71 52 21 29 55 47 31 45 42 67 42 38

Vasn TCAGTCCTTCACCTCTGG chr16 4640070 F 4 5 3 3 1 4 8 14 26 24 21 8 17


Supplemental Fig. 9. Validation of MSCC counts by Combined bisulfite

restriction analysis. (A) COBRA analysis of normal thymocytes (N),

MYC;Dnmt3bF/F and MYC;Dnmt3b-/- tumors in loci surrounding 21 HpaII sites.

PCR fragments were digested with a restriction enzyme (BstUI, TaqI*, or TaiI†)

and loaded onto a PAGE gel. CpG represents PCR fragment derived from

100% methylated human genomic DNA control. Undigested (U) and digested

(D) fragments correspond to unmethylated and methylated DNA, respectively.

Non-confirming COBRAs are colored in red. (B) MSCC data correlating to

counts assessed by COBRA in (A). Higher counts inversely correlate with the

methylation status of the HpaII site.


Th -/-F/F

MYC;Dnmt3b

Supplemental Fig. 10. Supervised hierarchical clustering of MSCC

samples based on count data. Supervised clustering was performed using

the most highly changed counts from MYC;Dnmt3bF/F and MYC;Dnmt3b-/-.

Spearman rank correlation was chosen for the similarity metric and average

linkage clustering of three normal thymus, and five MYC;Dnmt3bF/F and

MYC;Dnmt3b-/- tumors was conducted. Michael Eisen’s Gene Cluster program

was used for the clustering and Treeview to view the clusters.


A

Thymocytes

MYC;Dnmt3bF/F

MYC;Dnmt3b-/-

Thymocytes

MYC;Dnmt3bF/F

MYC;Dnmt3b-/-

Pnldc1 Promoter CpGs (4 clones)

Arhgef18 Promoter CpGs (6 clones)B

Supplemental Fig. 11. Methylation analysis of Pnldc1 and Arhgef18

promoters. (A) Bisulfite sequencing of the Pnldc1 promoter using genomic

DNA isolated from normal thymocytes, two MYC;Dnmt3bF/F and three

MYC;Dnmt3b-/- tumors. Each pie represents a CpG dinucleotide and each

wedge of pie represents the sequence of an individual allele. Black wedges

correspond to methylated CpG dinucleotides and white wedges represent

unmethylated CpGs. (B) Bisulfite sequencing of the Arhgef18 promoter using

DNA isolated from normal thymocytes, MYC;Dnmt3bF/F and MYC;Dnmt3b-/-

tumors. (C) qRT-PCR analysis of Pnldc1 in two averaged normal thymi (Th),

three MYC;Dnmt3bF/F and three MYC;Dnmt3b-/- tumors. (D) qRT-PCR analysis

of Arhgef18 in two averaged normal thymi (Th), two MYC;Dnmt3bF/F and three

MYC;Dnmt3b-/- tumors.

0

0.5

1

1.5

C

D

0

500

1000

1500

2000

2500 Pnldc1

Arhgef18

MYC;Dnmt3b-/-Th MYC;Dnmt3bF/F

MYC;Dnmt3b-/-Th MYC;Dnmt3bF/F

Re

lative

Exp

ressio

nR

ela

tive

Exp

ressio

n

0

200

300

100

5

10

15

20

Dnmt3bF/FN Dnmt3b-/-

Re

lative

exp

ressio

n

0

500

1000

1500

2000

0Dnmt3bF/FN Dnmt3b-/-

Pnldc1

0

4000

6000

2000

Rpl39l


25

50

75

100

0Re

lative

exp

ressio

n

Ass1

125

250

375

500

0

Lmo2

Dnmt3bF/FN Dnmt3b-/- Dnmt3bF/FN Dnmt3b-/- Dnmt3bF/FN Dnmt3b-/-

Cfb

0

4000

6000

2000

Re

lative

exp

ressio

n


0

1200

1800

600


Crb10 Wif1

Hlady_Supplementary_Fig_12R

ela

tive

exp

ressio

n

5

10

15

20

Dnmt3bF/FN Dnmt3b-/-0

S100a6

1

2

3

4

Dnmt3bF/FN Dnmt3b-/-0

Psma8

0

100

200

300

400

500


Syce1

Pkn3

Supplemental Fig. 12. Validation of microarray by qRT-PCR. qRT-PCR

shows the transcript levels of 11 genes in three MYC;Dnmt3b-/- (Dnmt3b-/-)

versus three MYC;Dnmt3bF/F (Dnmt3bF/F) tumors. An average of two normal

thymocytes (N) serves as a control. Averages +/- SEM is shown, P<0.05 for all

genes.

Supplemental Fig. 13. siRNA-mediated knockdown in MYC;Dnmt3b-/- cell

lines. (A) Dnmt3b-deficient cell lines were infected with non-targeting siRNA

controls (NT) or siRNA against Ment, Psma8, Rpl39l, Syce1, and Pnldc1. qRT-

PCR was performed to measure levels of transcript after two days of in vitro

incubation. Averages +/- SEM is shown, P<0.05. (B) An additional

MYC;Dnmt3b-/- cell line was treated with Ment siRNA to confirm the ability of

Ment to decrease cell growth.


0

20

40

60

80

100

120

Ment

Control

Rela

tive

Exp

ressio

n

A

B

Norm

alize

d C

ell C

ou

nts

0

20

40

60

80

100

120

140

Ment Psma8 Rpl39l Pnldc1

NT

siRNA

0

2

4

6

8

10

Locus A

U

D

TestesKidney

HeartLung

Thymus

Locus A 26 CpGs (7 clones)


A

B

C

2x105

Rela

tive

Exp

ressio

n

Supplemental Fig. 14. Methylation and expression patterns of Ment in

normal tissues. (A) Expression of Ment transcript levels in 14 normal mouse

organs based on qRT-PCR data. (B) Methylation readout of the Ment promoter

in normal mouse tissues by COBRA. Undigested (U) and digested (D)

fragments correspond to unmethylated and methylated DNA, respectively. (C)

In depth methylation analysis of five normal mouse tissues – thymus, testes,

kidney, heart, and lung by bisulfite sequencing. Each pie represents a CpG

dinucleotide and each wedge of pie represents the sequence of an individual

allele. Black wedges correspond to methylated CpG dinucleotides and white

wedges represent unmethylated CpGs.

Jeko-1

Jurkat

RAJI

REH

Akata

JY

JVM-2

Daudi

0

1

2

3

4

5

JVM-2 JY Jeko1 RAJI Jurkat Akata REH Daudi

P<0.05

JVM-2 JY

JeKo-1

RAJI

Jurkat

Akata

REH

Daudi

CpG


MENT

DNMT3B

Supplemental Fig. 15. Ment is a target of Dnmt3b-mediated methylation

in human lymphomas. (A) qRT-PCR analysis of MENT and DNMT3B

expression in human lymphoma cell lines. Statistically significant inverse

correlations were found using Spearman rank correlation (rs=-0.8 P=0.03). (B)

COBRA of MENT Locus II in human cell lines. (C) Bisulfite sequencing of

MENT Locus II in indicated cell lines.

A

B

C

D

U

Rela

tive E

xpre

ssio

n

Locus II

Locus II

Mantle Cell Lymphoma

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 26 CpG

D

U


Burkitt’s Lymphoma Diffuse Large B Cell Lymphoma

Supplemental Fig. 16. Methylation analysis of the MENT promoter in

primary human lymphomas. Methylation analysis of the MENT promoter by

COBRA in 26 human primary tumor samples from Burkitt’s Lymphoma

(samples 1-10), Diffuse Large B Cell Lymphoma (samples 11-20), and Mantle

Cell Lymphoma (samples 21-26). PCR fragments were digested with

restriction enzyme BstUI and loaded onto a PAGE gel. CpG represents a PCR

fragment derived from 100% methylated JURKAT genomic DNA control.

Undigested (U) and digested (D) fragments correspond to unmethylated and

methylated DNA, respectively.

0

1

2

3

4

5

6

7Dnmt1

0

1

2

3

4

5

6Dnmt3a

F/F F/F 3d+OHT 7d+OHT

Cre-ER

F/F F/F 3d+OHT 7d+OHT

Cre-ER


Supplemental Fig. 17. Acute loss of Dnmt3b in MYC;Dnmt3bF/F cells

leads to upregulation of Dnmt1 and Dnmt3a. MYC;Dnmt3bF/F cell lines

were infected with MSCV-EGFP-CreER constructs. Application of 4-

hydroxytamoxifen (OHT) allows Cre to enter the nucleus and conditionally

delete Dnmt3b. qRT-PCR was performed to measure levels of transcript of

Dnmt1 and Dnmt3a after three and seven days of incubation with OHT.

Averages +/- SEM is shown, P<0.05.

Rela

tive E

xpre

ssio

nR

ela

tive E

xpre

ssio

n

1.2

1.0

0.8

0.6

0.4

0.2

0Control shRNA-1 shRNA-2

Rela

tive E

xpre

ssio

n

P<0.05


Supplemental Fig. 18. Knockdown efficiency of Ment transcript levels by

qRT-PCR. Dnmt3b-deficient cell lines were infected with non-targeting shRNA

vector controls or one of two shRNA constructs to form stable cell lines. Cells

containing the shRNA vectors were selected for by addition of puromycin into

culture medium. qRT-PCR was performed to measure levels of transcript of

Ment after two days of in vitro selection. Averages +/- SEM is shown, P<0.05.

25 24

25 49

Contr

ol

Ment

EGFP

0 days 10 days


Supplemental Fig. 19. Up-regulation of Ment contributes to accelerated

cell growth in vitro. EGFP expression in unselected MYC;Dnmt3bF/F cells

infected with the MSCV-IRES-EGFP (control) or MSCV-myc-Ment-IRES-EGFP

(Ment) at plating (day 0) and after 10 days of in vitro growth. The percentage

of positive cells in the FACS profile is shown in the top right corner.

0

20

40

60

80

100

120

MENT

Control

Jurkat


Supplemental Fig. 20. Knockdown efficiency of MENT transcript levels

by qRT-PCR. A Jurkat cell line was infected with non-targeting siRNA (Control)

or siRNA designed against MENT. qRTPCR was performed to measure levels

of transcript of MENT after 48 days of in vitro incubation with siRNA. Averages

+/- SEM is shown, P<0.05.

Rela

tive

Exp

ressio

n

Documents

SUPPLEMENTAL TABLES Supplemental Table 2. Targets of … · 2014. 1. 30. · SUPPLEMENTAL TABLES Supplemental Table 2. Targets of Dnmt3b activity as determined by MSCC analysis. Methylation