Supplementary figure legend:
S Figure 1: Heat map of quantitative miRNA profiling of (A) EBV infected and (B) EBNA2 transfected U2932 cells:
A: Hierarchial clustering of U2932 EBVGFP clA and U2932 EBVGFP clB (EBNA2 negative and positive respectively) was performed analysing the microRNA that were found differentially expressed between the two groups. A red-green color scale depicts normalised miRNA expression levels in Ct values (Red: high, Green: low).
B: The comparison of principally altered miRs in EBNA2 overexpressed U2932 EBNA2 cl2 vs. the parental cell line U2932 MPA vector. Heatmap shows the average change in miRNA expression among three independent samples (n=3). A red-green color scale depicts normalised miRNA expression levels in Ct values (Red: high, Green: low).
S Figure 2: pAKT expression and the effect of miR-21 knock-down in EBV infected and EBNA2 expressing BL41 and Bjab Cells:
A and C: pAKT and total AKT expression was analyzed by immunoblotting in BJAB, EBV infected BJAB EBVGFP cl.4, EBNA2 expressing BJAB K3, BL41 infected with EBV (BL41 E95C) and BL41 transfected with EBNA2. The role of miR-21 in the regulation of pAKT was further studied by knocking down miR-21 by transfecting antisense LNA miR-21 oligonucleotide. B and D: Expression of miR-21 as analyzed by taqman q-PCR in the corresponding cells in A and C. The figure represents mean of three experiments, each performed in triplicates. The values are normalized to an internal U6 RNA control.
S Figure 3: Primary miR-21 transcript analysis in EBV infected and EBNA2 transfected U2932 by qRT-PCR:
Primer pairs were chosen from sites flanking the Drosha-DGCR8 cleavage sites. Real time qRT-PCR was performed in triplicates. The qPCR were normalized against an internal RPL32 RNA expression. Two different RNA extraction from all cell lines were tested for pri-miR-21 expression. A: EBV infected U2932 EBVGFP cl-A and cl-B were compared to the uninfected parental cell line for pri-miR-21 levels. B: EBNA2 transfected clones of U2932 were compared to the parental and the vector transfectant. The expression of pri-miR-21 in the vector transfected U2932 cells was set at 1. Expression of EBNA2 as detected by immunoblotting is shown below each panel.
S figure 4: miR-21 promoter activity in EBNA-2 transfected U2932 cells:
miR-21 promoter linked to luciferase reporter was transiently transfected into U2932 MPA vector and EBNA2 transfected clones. After 48 hours, cells were harvested and tested for reporter activity using the dual luciferase assay kit.
S Figure 5: miR-146a promoter activity in U2932 cells expressing LMP1 and EBNA2: miR-146a promoter linked to luciferase reporter was transiently transfected into MPA vector, LMP1 and EBNA2 expressing U2932 cells. After 48 hours, cells were harvested and tested for reporter activity using the dual luciferase assay kit. The mean shows ± S.D., n=3.
S Figure 6: Apoptosis, Cell cycle and proliferation in EBNA2 expressing U2932 cells and the effect of miR-21 knockdown:
A: Expression of BCL2 and BCLxS as analyzed by immunoblotting.
B: 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).
C: p21, p27 and CDK4 expression as evaluated by immunoblotting.
D: Effect of miR-21 knockdown on p21 expression.
E: Cell cycle distribution of EBNA2 epxressing U2932 cells compared with controls.
F: In vitro proliferation and the effect of miR-21 knockdown in U2932 EBNA2 cells.
G: Agarose cloning efficiency of U2932 EBNA2 expressors and the effect of miR-21 knockdown in these cells compared to controls.