Supporting Information
Yustein et al. 10.1073/pnas.0901230107
SI Materials and Methods
The Gamma-secretase inhibitors Factor X and DAPT was purchased from Sigma-Aldrich and dissolved in DMSO for tissue
culture experiments or a 10% ethanol/corn oil solution for s.c.
injections into SCID mice. Primers for real-time RT-PCR were
obtained from IDT Technologies and resuspended in nucleasefree water. Jagged2 antibody was purchased from R&D Biosciences, and c-Myc (9E10) mouse monoclonal antibody was
used. Actin or tubulin mouse monoclonal antibody was used.
Ready-Gel Tris-Glycine acrylamide gels (Bio-Rad) were used for
electrophoresis. Tetracycline and beta-estradiol were purchased
from Sigma-Aldrich.
Cell Lines and Tissue Culture. Burkitt lymphoma cell lines CA46,
Ramos, and Raji were from ATCC. P493-6 human B-cell lymphoma cell line was cultured as described (1). Growth assays were
with 5,000 cells/well in a 96-well plate. Ten microliters per well of
CCK-8 tetrazolium-based colorimetric reagent (Dojindo, Inc.)
was added each day, 4 h before measurement at 450 nm. Daily
values were performed in triplicates and averaged. Tetracycline
(0.1 μg/mL) was applied for 72 h to reduce c-Myc. Tetracycline
release experiments were performed by washing P493-6 cells
with 1× PBS twice and then placing cells in media without tetracycline. Beta-estradiol (1 μM final concentration) was added
and changed every 3 days. Human Jagged2-GFP/MSCV construct was a gift of Nadia Carlesso (Indiana University, Bloomington, IN). Hypoxia experiments were performed as follows:
Cells were maintained in standard media with 10% FCS and 1%
penicillin/streptomycin at 37 °C in 5% CO2 and 95% air. Hypoxic
conditions were created by flushing 5% CO2 and 95% N2
through a chamber at 37 °C, until an atmosphere containing 1%
O2 was achieved, and measured with oxygen meter.
Immunoblotting. Whole-cell lysates were obtained by lysing in
MPER (Pierce) lysis buffer and quantified using BSA protein
quantification assay (Pierce). Twenty micrograms of whole-cell
lysate were loaded per lane for each blot, which was developed
with ECL Western Blot reagent (Amersham). β-actin or -tubulin
was used as loading control. Anti-NICD antibody was obtained
from Cell Signaling and used at 1:1,000 dilution.
Immunohistochemistry. Tumor hypoxic areas were detected by pimonidazole hydrochloride (Hypoxyprobe) from Natural Pharmacia International. A total of 1.5 mg Hypoxyprobe diluted in
150 μL of 0.9% saline was given via i.p. injection 1 h before tumors
were rapidly harvested and fixed in 10% neutral formalin buffer.
Aqua DePar and Bord Decloaker RTU (Biocare Medical) were
used according to the two-step deparaffinization and heat retrieval
protocol of the manufacturer. Protein adducts of reductively activated pimonidazole were detected by rabbit anti-hypoxyprobe
antibody (1:100). The immunofluorescence reaction was visualized by Texas Red goat anti-rabbit antibody (1:200). For Jagged2 detection, goat anti-Jagged2 (1:400; Santa Cruz) and donkey
polyclonal to goat FITC conjugated (1:200; Abcam) were used as
primary and secondary antibodies after 1 h blocking with 10%
donkey serum. Samples were analyzed under Axiovert 200 (Zeiss)
fluorescence microscope at 10× magnification. Tissue microarrays
were stained with the goat anti-Jagged2 antibody (1:400; Santa
Cruz) using Dako Biotin Blocking System. Human samples were
used with approval from the Johns Hopkins University School of
Medicine Institutional Review Board.
Yustein et al. www.pnas.org/cgi/content/short/0901230107
RNA Isolation and Real-Time PCR. RNA from P493-6 cells was isolated
as described (1). cDNA was prepared using TaqMan Reverse Transcription Reagents (Roche, Applied Biosystems). Quantitative realtime PCR was performed using the ABI 7500 sequence detection
system. A predeveloped probe and primers specific to 18S rRNA levels
were used for normalization. All PCRs were performed in triplicate.
Primers used for real-time PCR are listed by gene name (forward
prime sequence; reverse primer sequence): Jagged2 (5′ GATACCA
CCCCGAATGAGGAG 3′; 5′ GGGTTGATCATGCCGGC 3′);
PKC iota (5′ TGAGCAGGCATCCAATCATC 3′; 5′ TGAAAGCAAGAATGCAGCCC 3′); ALDH5A1 (5′ TGTGCAATGTCA
CCCAGGAC 3′; 5′ GCCCGAAAGTCTCTTCATGAGT 3′); PRR5
(5′ TGCTGGCCAAGAACCCTG 3′; 5′ GCAGAGGCGTGTTGT
AGCTCT 3′); TAF4B (5′ ATGCTGTGAACTTGATCTCCCA 3′;
5′GAAGGCCTCGTAGTCGTTCCT 3′); MLK4 (5′ ACAGATTTT
GGGTTGGCGAG 3′; 5′ TGTGCTCATTTTGGTGGTCCT 3′);
ACY1L2 (5′TTTTATGGCCCACCCATCAC 3′; 5′GCCATATC
TGGTAGATAAGCAGCA 3′); SCAMP1 (5′ ATCTTCGGATGC
TTGGCTTG 3′; 5′ ATCAACCGCTCTTGCAGAATC 3′); CSRP2
(5′ GAGGTGCAGTGTGATGGCAG 3′; 5′ CATGCAGAGAA
AGCAGCAGC 3′); ASB2 (5′ATCATGTTCGCCATGAAGTGC
3′; 5′ CAGGTCCATGAGGAACTTGAGC 3′); HES-1 (5′AAGAAAGATAGCTCGCGGCA 3′; 5′ TTTCCAGAATGTCCGCC
TTC 3′); NRARP (5′ TGCACCAGTCGGTCATCG 3′; 5′ TTGACC
AGCAGCTTCACGAG 3′); Notch1 (5′ TTTACTGCGACGTG
CCCAG 3′; 5′ ACGTCAACACCTTGTCGCTG 3′).
Chromatin Immunoprecipitation (ChIP) Assay. This assay was performed
as described previously (1). Real-time PCR using SYBR Green
(Applied Biosystems) was performed per manufacturer’s protocol.
The primers used for analysis of the Myc binding sites within the
JAG2 promoter region as well as within intron 2 are in Table S1.
RNA Interference. Jagged2 SMARTpool siRNA was purchased
from Dharmacon, Inc. 3 × 106 P493-6 cells were electroporated
(240 V and 1,500 μF in 4-mm cuvettetes) using Bio-Rad gene
pulser with siRNA for final concentration of 100 nM. After
electroporation, cells were immediately placed into full media
and evaluated at the indicated times.
In Vivo Tumorigenesis and γ-Secretase Inhibition. SCID mice were
injected with 25 million P493-6 cells each on day 0 and then either
treated with s.c. injections of DAPT of ≈60 mg/kg per dose or
control solvent 3 times a week for 2 weeks starting on day +3. Five
mice were used for each condition, and the experiment was performed twice with similar results. Tumors were measured using
standard calipers, and volumes were determined using the following formula: length × width × width × 0.52.
Microarray Analysis. After isolation and purification of RNA from
P493-6 cells under the indicated conditions, 10–15 μg was supplied for microarray analysis at the Johns Hopkins Microarray
Core Facility. The RNA samples were analyzed with Affymetrix
GeneChip Human 133 2.0 Arrays. Quality of the microarray
experiment was assessed with affyPLM and Affy, two bioconductor packages for statistical analysis of microarray data.
Gene Expression Cohort for Clustering Analysis. Gene expression
dataset generated on Affymetrix platforms of a cohort of 101
multiple myeloma (MM), 22 monoclonal gammopathy of
undetermined significance (MGUS), 14 Waldenstrom macroglobulinemia (WM), 7 chronic lymphocytic leukemia (CLL), 8
Burkitt’s and 7 atypical Burkitt’s lymphoma, 6 normal donors of
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peripheral blood B cells (BC), and 15 normal donors of plasma
cells (PC) were obtained from the Gene Expression Omnibus
database (accession nos. GSE6477 and GSE4475).
Gene Expression Analysis for Gene Clustering. Probe level intensity was
generated using MAS5.0. Data are median centered and analyzed
in Genespring GX 7.3.1. The genes that have a present call in less
than 30% of samples are filtered out. The remaining genes are
subjected to fold-change analysis between the HIGH Myc and NO
Myc cell lines. Genes that are 5-fold different between the HIGH
Myc and NO Myc cell lines are used to cluster the samples (cell
lines with different Myc expression and other B-cell and plasma cell
tumors). Clustering was performed by hierarchical clustering using
Pearson correlation coefficient and centroid linkage.
Array-Based Nuclear Run-On. Isolated nuclei were added to NRO Re-
action Buffer (300 mM KCl, 10 mM MgCl2, 2 mM of cold rATP,
rCTP, rGTP, and 1 mM biotin-modified UTP, ribonuclease inhibitor
such as Invitrogen RNaseOUT 100 U) and incubated at 28 °C for
25–30 min. RNase-free DNase I (100 U; Roche) was added and the
mixture incubated 10 min at 37 °C followed by 15 μL 3:1 of Proteinase K (20 mg/mL): 10% SDS; incubate 10 min at 37 °C. RNA
was isolated using the Qiagen RNeasy RNA Isolation Kit. Biotinylated RNA was isolated using Dynabeads M-280 Streptavidin.
Using the magnet stand, RNA-bound Dynabeads were sequentially
washed with RNase-free H2O six times.
cDNA synthesis was performed using Ambion TotalPrep RNA
Amplification Kit with the washed RNA-bound beads in T7N6
Primer Solution. cDNA was purified with Ambion TotalPrep
RNA Amplification Kit.
Array Analysis. For Illumina arrays, biotin-labeled cRNA was
combined with hybridization buffer + formamide and hybridized
(16 h) at 55 °C to Illumina’s Sentrix HumanRef-8 Expression
BeadChips (Illumina).
1. Iso T, Kedes L, Hamamori Y (2003) HES and HERP families: Multiple effectors of the
Notch signaling pathway. J Cell Physiol 194:237–255.
Fig. S1. Microarray validation of several genes induced only upon ectopic Myc expression via qPCR. Primers used for qPCR can be found in Table S1. Cells were
treated with tetracycline for 48 h, estradiol/Tet for 48 h, and untreated.
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Fig. S2. Myc and Jagged2 protein expression in human B cells and Burkitt lymphoma cell lines. (A) Immunoblots of cell lysates from human CB33 lymphoblastoid cells as compared with CB33 overexpressing Myc and P493-6 B cells with HIGH Myc. (B) Immunoblots of cell lysates from human Burkitt lymphoma cell
lines CA46, Ramos, and Raji. (C) Expression of JAG2 mRNA and nuclear run-on rate in P493-6 cells as a function of time after tetracycline washout. JAG2 mRNA
levels normalized to 18S control were measured by real-time PCR at time points after tetracycline removal to induce Myc in P493-6 cells. (D) Nuclear run-on
(NRO) and total (Total) RNA levels measured by Illumina bead microarray in triplicate biological experiments at time points after tetracycline removal to induce
Myc in P493-6 cells.
Fig. S3. Correlation between Myc chromosomal translocation and mRNA levels of Myc and JAG2 in primary human lymphomas. (A) Box plots from the
Oncomine database (https://www.oncomine.org/resource/login.html) depicting the statistically signification differential expression of Myc and JAG2 in lymphomas that bear Myc chromosomal translocations (n = 59) versus those lacking Myc rearrangements (n = 141). P values from t test are shown beneath the
panels. (B) Expression of Jagged2 protein in human medulloblastoma tissue microarrays and P493-6 xenograft tumors. Micrographs of representative tissue
sections on microarrays of anaplastic (HIGH Myc) and classic medulloblastomas. (Bottom) Distribution of Jagged2 staining intensity among cases of classic and
anaplastic medulloblastomas. (C) Immunofluorescent micrographs of P493-6 tumors labeled in vivo with pimonidazole to detect hypoxia (red) and Jagged2
expression (green).
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Fig. S4. (A) Expression of hypoxia-induced genes and Notch signaling target genes in P493 and P493 JAG2 cells under normoxic and hypoxic conditions.
Relative expression of mRNA levels for the indicated genes was determined by real-time PCR (primer sequences shown in Table S1) in various cell samples. (B)
Expression of NRARP and Deltex in cells retrieved from P493 or P493 JAG2 xenograft tumors. Levels of NRARP and Deltex were determined by real-time PCR
normalized to 18S control.
Other Supporting Information Files
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