American Society of Hematology

Figure 5

$Figure 5. Blockade of VLA-4/VCAM-1 signaling impairs NF-κB activation and transcription of NF-κB downstream target genes. Normal BM-MSCs were cocultured for 24 hours with (A) REH or (B) OCI-AML3 cells that were either untreated or preincubated with VLA-4 bAb for 1 hour. When indicated, 200 ng/mL of IL1RA was added to the coculture medium. BM-MSCs were cultured alone (monocultured) as controls. After separating the cells (as indicated in Methods), total RNA from BM-MSCs and leukemic cells was extracted. (A-B) Expression of selected NF-κB target genes in cocultured BM-MSCs was determined by qRT-PCR. Bars represent qRT-PCR data from triplicate samples, and results are expressed as fold difference expression (±SEM) in each coculture condition vs the monocultured BM-MSCs. (C-D) Total RNA from (C) REH and (D) OCI-AML3 ells (cocultured with MSCs in experiments shown in A and B, respectively) was extracted, and IL-1α and IL-1 β mRNA expression levels were quantified by qRT-PCR. Bars represent qRT-PCR data from triplicate samples, and results are expressed as fold difference expression (±SEM) in each coculture condition vs the monocultured OCI-AML3 cells. (E) Western blot analysis of cytosolic (CF) and nuclear (NF) fractions of lysates from REH and OCI-AML3 cultured alone (−) or cocultured with BM-MSC (+) for 1 hour. (F) Western blot analysis of cytosolic (CF) and nuclear (NF) fractions of BM-MSC lysates cultured alone (−) or cocultured with OCI-AML3 or REH cells for 1 hour. Leukemic cells were preincubated with VLA-4 bAb when indicated (+) before coculture. Each well corresponds to 5 µg of total protein. Membranes were probed with rabbit monoclonal anti-p65, mouse monoclonal anti-PARP1 (nuclear fraction loading control), and mouse monoclonal anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (cytosolic fraction loading control).$

Blockade of VLA-4/VCAM-1 signaling impairs NF-κB activation and transcription of NF-κB downstream target genes. Normal BM-MSCs were cocultured for 24 hours with (A) REH or (B) OCI-AML3 cells that were either untreated or preincubated with VLA-4 bAb for 1 hour. When indicated, 200 ng/mL of IL1RA was added to the coculture medium. BM-MSCs were cultured alone (monocultured) as controls. After separating the cells (as indicated in Methods), total RNA from BM-MSCs and leukemic cells was extracted. (A-B) Expression of selected NF-κB target genes in cocultured BM-MSCs was determined by qRT-PCR. Bars represent qRT-PCR data from triplicate samples, and results are expressed as fold difference expression (±SEM) in each coculture condition vs the monocultured BM-MSCs. (C-D) Total RNA from (C) REH and (D) OCI-AML3 ells (cocultured with MSCs in experiments shown in A and B, respectively) was extracted, and IL-1α and IL-1 β mRNA expression levels were quantified by qRT-PCR. Bars represent qRT-PCR data from triplicate samples, and results are expressed as fold difference expression (±SEM) in each coculture condition vs the monocultured OCI-AML3 cells. (E) Western blot analysis of cytosolic (CF) and nuclear (NF) fractions of lysates from REH and OCI-AML3 cultured alone (−) or cocultured with BM-MSC (+) for 1 hour. (F) Western blot analysis of cytosolic (CF) and nuclear (NF) fractions of BM-MSC lysates cultured alone (−) or cocultured with OCI-AML3 or REH cells for 1 hour. Leukemic cells were preincubated with VLA-4 bAb when indicated (+) before coculture. Each well corresponds to 5 µg of total protein. Membranes were probed with rabbit monoclonal anti-p65, mouse monoclonal anti-PARP1 (nuclear fraction loading control), and mouse monoclonal anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (cytosolic fraction loading control).

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