Coculture with leukemia cells induces NF-κB activation in BM-MSCs. (A) A group of array-identified upregulated genes in cocultured BM-MSCs was validated by qRT-PCR. The bars represent qRT-PCR data from 3 independent experiments, and results are expressed as fold difference expression (±SEM) in the cocultured BM-MSCs vs the monocultured BM-MSCs. (B) A total of 5 ALL and 7 AML patient samples were independently cocultured with BM-MSCs and processed as in A. Data from qRT-PCR analysis are expressed as fold difference expression in cocultured BM-MSC vs the monocultured BM-MSCs. Mean expression value for each gene is shown as a bar. (C) qRT-PCR analysis showing expression levels of a selected group of NF-κB target genes in AML-MSCs (n = 12) compared with N-MSCs (n = 8). A total of 12 AML-derived and 8 normal donor-derived primary MSC cultures were analyzed. Bars represent the mean value (±SEM) Log2 expression levels relative to ABL (housekeeping control) expression levels. (D) Western blot analysis of cytosolic (CF) and nuclear (NF) fractions of lysates from BM-MSCs cultured alone (−) or cocultured with REH or OCI-AML3 cells for 1 hour. 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 (cytosolic fraction loading control). (E) Immunofluorescence staining for p65/RelA showing p65 translocation into BM-MSC nuclei on interaction with leukemia cells in coculture conditions. BM-MSCs were cultured alone (monoculture) or cocultured with REH cells for 24 hours and then fixed with 4% paraformaldehyde (PFA). As controls, BM-MSCs cultured alone were treated with vehicle or TNFα (20 ng/mL) for 30 minutes. Nuclei were counterstained with DAPI. Scale bar, 10 µm. Arrows point at absence (monoculture panel) or presence (coculture panel) of p65 in nuclei.