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Blood, Vol. 112, Issue 9, 3798-3806, November 1, 2008
IRF-4 functions as a tumor suppressor in early B-cell development
Blood Acquaviva et al.
112: 3798
Supplemental materials for: Acquaviva et al
Files in this Data Supplement:
- Figure S1. Imatinib treatment of BCR/ABL B-ALL cell culture results in increased IRF-4 mRNA levels (JPG, 36.2 KB)
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A fraction of imatinib-treated and untreated BCR/ABL B-ALL sample prepared for analysis in Figure 1 were used to detect the IRF-4 transcripts using real time RT-PCR analysis in triplicate.
- Figure S2. Imatinib treatment inhibits cell cycle progression and increases apoptosis in BCR/ABL B-ALL cells (JPG, 71.3 KB)
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Cell cycle analysis of imatinib-treated cells at 36h shows a statistically significant accumulation of cells in G1/G0 at 5µM (P=2.8 × 10−5) and 10µM (P=5 × 10−4) imatinib (A&B) and a significant increase in the percentage of apoptotic cells at 5µM (P=3 × 10−4) and 10µM (P=0.001) compared to untreated cells (A&B). Analysis of BrdU incorporation and 7-Amino-actinomycin D (7-AAD) levels allowed distinction of cell cycle phases G1/G0, G2/M, S and dying/dead cells (Ap). Percentage of cells in each phase is indicated within the gate. P values obtained from students unpaired t test.
- Figure S3. Expression of BCR/ABL, myc-tagged IRF-4, myc-tagged IRF-8 and endogenous IRF-4 (JPG, 71.2 KB)
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(A) Expression of BCR/ABL, myc-tagged IRF-4, myc-tagged IRF-8 in the E2AGFP pro–B-cell line infected with MSCV-BCR/ABL-GFP+Neo, MSCV-BCR/ABL-GFP+IRF-4, or MSCV-BCR/ABL-GFP+IRF-8 as detected by immunoblotting with an anti-ABL monoclonal antibody (Ab-3) (top panel) and anti-myc tag antibody (9E10) (second panel from top). (B) Comparison the amounts of ectopically expressed IRF-4 in MSCV-BCR/ABL-GFP+IRF-4–infected E2AGFP pro-B cells to those of endogenous IRF-4 in imatinib-treated BCR/ABL transformed E2AGFP cells as detected by immunoblotting with an anti–IRF-4 polyclonal antibody. An anti-dynamin monoclonal antibody was used to detect dynamin levels as a loading control. Densitometry analysis using NIH Image J software was used to determine the relative expression levels of ectopic and endogenous IRF-4. (C) Expression of myc-tagged IRF-4 and myc-tagged IRF-8 in BCR/ABL expressing B-ALL cells transduced with MSCV-RFP, MSCV-RFP+IRF-4, or MSCV-RFP+IRF-8 as detected by immunoblotting with anti-myc tag antibody (9E10) (bottom panel). Anti-dynamin blot was used as a loading control. (D) Expression of IRF-4 (endogenous or ectopic + endogenous) in BCR/ABL expressing B-ALL cells transduced with MSCV-RFP, MSCV-RFP+IRF-4 or MSCV-RFP+IRF-8 and imatinib-treated B-ALL cells as detected by immunoblotting with the anti–IRF-4 antibody. The anti-dynamin blot was used as a loading control. Densitometry analysis using NIH Image J software was used to determine the relative expression levels of ectopic and endogenous IRF-4.
- Figure S4. Growth and mRNA levels of cell cycle regulators in BCR/ABL B-ALL cell lines with IRF-4 WT; IRF-4 Het or IRF-4 KO (JPG, 74.3 KB)
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(A) 105 cells for each cell lines were plated in triplicate and grown for three days. Cells were counted by trypan blue exclusion. BCR/ABL expressing IRF-4 KO B-ALL cells (#9) grew faster than that of BCR/ABL expressing IRF-4 WT B-ALL cells (#8, P=0.01) and BCR/ABL expressing IRF-4 Het B-ALL cells (#23, P=0.002). (B–E) Real time RT-PCR analysis was performed to determine the mRNA levels of cell cycle inhibitors including Cdkn1b, Cdkn1c, Cdkn2a and Cdkn2b. The housekeeping gene β-2-microglobin (B2m) was used as the internal control. There were no significant differences in expression of Cdkn1b, Cdkn1c, and Cdkn2a mRNA among the three cell lines (B-D). Cdkn2b mRNA levels was significantly lower in BCR/ABL expressing IRF-4 KO B-ALL cell line compared with either BCR/ABL expressing IRF-4 WT or Het B-ALL cell lines (P<0.05) (E).
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