Supplementary material and methods:
Infection with a recombinant EBV strain:
The supernatant containing recombinant EBV was used to infect EBV negative U2932
cells. The viral infection was carried out for two hours at 37 o C, with intermittent shaking.
The cells were washed and resuspended in complete medium for 48 hours before
selection in 1 mg/ml of G418. Three weeks later, the GFP positive and G418 resistant
clones were propagated.
Immunoblotting:
EBNA2 expression was verified by using a monoclonal antibody, PE2 (Kindly provided by
Dr. Martin Rowe, Birmingham University Medical School). The expression of LMP1 was
detected with S12 monoclonal antibodies (a kind gift of Dr. David Thorley-Lawson, Tufts
-actin antibodies were purchased from Sigma. AKT, p-AKT
and IRAK1 antibodies were purchased from Cell Signaling. Cell cycle proteins related
antibodies are as follows: Cell cycle antibodies: Kip/p27 (BD Transduction Laboratories),
Cdk4 and Cip/p21 are fro mSanta Cruz Biotechnologies. Apoptosis related protein
antibodies are: BCL2 (DAKO) and BCL-xS (Thermo Scientific Pierce Antibodies). The
working conditions of all antibodies were those indicated by the respective providers. The
chemiluminescence kit (Amersham) was employed to visualize the proteins.
Locked nucleic acid based miR microarray:
Each slide was placed in a slide chamber (Ambion Cat.#10040) and hybridized in a water
bath for 16 hrs at 60°C. Low stringency and high stringency washes were carried out and
the microarrays dried following the Exiqon washing manufacturer’s protocol. A ScanArray
Lite Microarray Scanner (Packard Bioscience) was used to acquire images, and GenePix
Pro 6.0 software was used to quantify hybridization signals. Absent and marginal spots
were flagged automatically by the software and subsequently each slide was inspected
manually. Microarray images were processed and analyzed using GenePix Pro 6.0, Excel
and TIGR Multiexperiment viewer version 4.0 software. The data were pre-processed and
normalized using spike-in capture probes spotted onto slides and different positive control
capture probes. The resulting generalized log2 values were used in further data analysis.
Northern blotting:
After electrophoresis on urea-acrylamide gels, the RNA was transferred to hybond nylon
filters at 10 volts overnight at 40 C. After the U.V. cross-linking, the filters were
prehybridized in 6 X SSPE, 5x DENHARDT and 0,5 % SDS solution. Ten picomoles
probes were radiolabelled using P32 ATP by standard reaction at 370 C. Microspin G25
columns (Amersham) were used to separate radiolabelled probe from unincorporated
ATP. The prehybridization and hybridization were performed in the same buffer, at 37 0 C
overnight. The filters were washed once in pre-warmed washing solution containing 6x
SSPE and exposed in cassettes containing phosphor screen overnight. The signals were
read by Amersham Typhoon 9200 phosphoimager and densitometry was performed with
IMAGEQUANT software (Amersham). The filters were stripped in a solution containing 0,2
X SSPE and 0,2 % SDS for 10 minutes at 95 0 C and subsequently rehybridized with
another probe.
Quantitative RT-PCR:
The cDNA synthesis for mature miR-21 and miR-146a was performed according to the
manufacturer’s instructions (Applied Biosystem, Cat#4366597). For the verification of primiR-21 and pri-miR-146a expression the reverse transcription was performed as follows:
to each DNAse treated RNA sample, 33 ng random hexamers, 2 l of 10mM dNTPs were
added and incubated at 70° C for 5 min. The following reaction components were added:
12 l of 5x 1st strand buffer (Superscript III reverse transcriptase, Invitrogen), 1,44 l
RNAsin (Promega) and 17,5 l of DEPC water. The final volume of 60 l was split into
2x30 l aliquots. To one aliquot, 1 l (200 units) of superscript III reverse Transcriptase
(Invitrogen) was added and to the other, 1 l of DEPC water to control for genomic DNA
contamination. The samples were then incubated for 10 minutes at 25° C, 50 min at 37° C
and 15 min at 70° C. Subsequently, each cDNA sample was split into three and used for
qRT-PCR. The mature miRs and IRAK1 expression was evaluated by employing specific
Taqman primers for miR-21, miR-146a and IRAK1 (Applied Biosystem HSA-miR-21
Cat.#373090, HSA-miR-146a Cat.# 4427975 and IRAK1 Cat.# 4331182). RNU6b
(Cat.#4373381) and GAPDH (Cat. #4326317) were used as housekeeping genes for miRs
and IRAK1 expression respectively. The Q-PCR reaction was performed using SensiMix
dT kit (Quantace), SYBR green (in case of pri-miR-21, pri-miR-146a, interferon A2 and
A4), 8 mM MgCl2 and 330 nM of each forward and reverse primer on a real time PCR
system 7300 from Applied Biosystem. For pri-miR-21 and pri-miR-146a amplification, the
primers were selected from regions flanking the Drosha cleavage sites. For normalization,
ribosomal protein L32 (RPL32) expression was studied. The primers sequences and PCR
conditions used are described in table 1. The relative amount of pri-miR-21 and pri-miR146a was calculated by comparative Ct (threshold cycle) method by subtracting the
average Ct value for RPL32 from the average Ct value for each pri-miR (Ct). Next, the
Ct values were calculated by subtracting the Ct value of the common calibrator
(U2932 or U2932 MPA vector) from the Ct of each sample. Finally, the relative
expression was defined as 2-Ct . The range of expression levels was determined by
calculating the standard deviation of the 2-Ct of three independent experiments, each
starting from a new RNA extraction.
For type I IFNs that are devoid of introns in their genes, PCR products used for the
standard curves have been quantified against human genomic DNA, thus allowing to
compare the level of expression of type I IFN mRNAs between each other. The type I IFN
primer pairs for IFNA2 and IFNA4 have been previously described (Coccia et al, Eur J
Immunol. 2004 Mar;34(3):796-805). The values of IFNA2 and IFNA4 are represented as 2Ct
since the levels of type I interferon expression in the calibrator sample (U2932 and
U2932 MPA vector) was below detection.
miR-146a and miR-21 luciferase promoter activity:
The transfection efficiency was measured by calculating renilla expression ratio between
the transfected and the untransfected cells. Firefly and Renilla activities were measured
using the Dual luciferase assay kit (Promega) by a luminometer (Perkin Elmer, 1420,
Multilabel counter).
For miR-21 luciferase promoter activity, U2932 EBNA2 cl-1 and cl-2 were transfected with
10 µg of miR-21 luciferase promoter (a kind gift from Prof. Hideo Iba, University of Tokyo).
The experimental conditions are the same as miR-146a
promoter luciferase assay,
described in the material and method section in the main text.
Quantitative miRNA expression profiling:
Expression profiling of 754 miRNAs was carried out on RNA samples according to Applied
Biosystems protocols. Briefly, reverse transcription was performed with 450ng total RNA
using Megaplex RT stem loop primers, Multiscribe Reverse Transcriptase, RNase inhibitor
and 100nM deoxynucleotide triphosphates (dNTPs) (reagents from Applied Biosystems,
Life Technologies). The multlplexed RT reaction was performed according to
manufacturer’s instructions. Quantitative real-time RT-PCR was done utilizing pre-printed
Taqman low density assay (TLDA) microfluidic cards (Human Card A v2 and Human Card
B v3, format 384 each). Each card set contained MGB labelled probes specific to mature
miRNAs plus endogenous small nucleolar RNAs (MammU6, RNU44, RNU48, and
U6snRNA) for data normalization and relative quantification. Pre-processing of raw TLDA
data files consisted of threshold and baseline corrections for each sample, with each
amplification plot assessed to confirm that the threshold cycle (Ct) value corresponded
with the midpoint of logarithmic amplification (SDS 2.3, Life Technologies).
Statistical Analysis: MicroRNA statistical analysis was carried out using the Wilcoxon
signed-rank test using StatMiner (Integromics) software. Hierarchial clustering analysis
was performed on the miRNA, representing the Ct values in linear scale. The comparative
threshold cycle method was used to calculate the relative miRNA expression. The
reproducibility of the assay was evaluated by considering three different RNA preparations
from all the samples analysed. Dendrograms represent complete linkage clustering (by
Euclidean distance) between sample subsets. The significantly modulated microRNAs
were defined as a P value ≤0.05.
Apoptosis and cell cycle:
Evaluation of the early (Annexin V+/PE-) and late (Annexin V+/PE+) apoptotic population
was performed with PE Annexin V Apoptosis Detection kit (BD Pharmingen) following the
manufacturer’s instructions.
Cell cycle distribution study was carried out with 1,5x10 6 cells washed twice in ice cold
PBS and incubated for 30 minutes at 4°C in 1ml of fixation/permeabilization solution
(eBioscience). Fixed cells were washed two times in the permeabilization buffer 1x
(eBioscience) and then stained intracellularly with 100 µl of 7AAD solution (25µg/ml 7AAD
(BD Pharmigen), RNAse 40µg/ml in PBS) for 30 minutes.
Apoptosis and cell cycle were analyzed with a BD Biosciences CellQuest software.
In vitro growth kinetics:
The cells were seeded at 2x10 5 per ml and re-seeded at the original concentration on the
fourth day, for two successive passages. The cells were counted daily and the viability was
checked by Trypan blue exclusion test.
Agarose cloning:
Peripheral blood mononuclear cells (PBMCs) from healthy donors were stimulated with
phytoaemagglutinin 1µg/ml for 1 hour at 37°C and irradiated at 3000 rads. One and a half
million PBMCs were mixed with 0,45% (w/v) SeaPlaque agarose (Cambrex Bio Science
Rockland, Inc) heated and diluted with a complete medium (RPMI1640, 10% FBS). Three
milliliters of the above cell suspension (feeder layer) were distributed in a 35x10 mm Petri
dishes. Following solidification the feeder layers were incubated at 37°C in a 5% CO2
atmosphere for 24 hours. Above the feeder layer, we poured 3 ml of a cell solution
containing1000 cells of each cell line mixed with 0,35% (w/v) agarose in a complete
medium. Colonies, determined as a group of more than 30 cells, were counted after 8 to
10 days of culture at 37°C. Each cloning experiment was performed three times and every
cell line was plated in triplicates. The results of the cloning efficiency are given as mean
and standard error of counted colonies in three different experiments.