The effects of overexpression of IκBα super repressor (IκBα-SR) in BM-MSCs. (A) Immunofluorescence staining for p65/RelA in BM-MSCs transduced with either empty control (MSC-Control) or IκBα-SR virus (MSC-IκBα-SR). p65/RelA nuclear translocation was determined by confocal microscopy analysis in cells treated with dimethylsulfoxide (vehicle) or TNFα (20 ng/mL) for 30 minutes before fixation with 4% PFA. Approximately 50 fields with 2 to 3 cells per field were analyzed per condition. The nuclei were counterstained with DAPI. Scale bar, 10 µm. (B) MSC-Control or MSC-IκBα-SR cells were cultured alone (monoculture) or cocultured with REH cells for 48 hours as indicated. Total RNA was extracted, and qRT-PCR was carried out to detect the expression of a selected group of NF-κB target genes. Results of 3 independent experiments are expressed as the mean fold difference expression (±SEM) in different culture conditions over the expression levels in the monocultured MSC-Control. (C) REH cells were cultured alone or cocultured with either MSC-Control or MSC-IκBα-SR and then treated with VCR for 72 hours. As described for Fig. 2, (left) the percentage of apoptotic cells and (right) absolute number of viable cells were assessed by flow cytometry using annexin V⁺/DAPI⁺ staining and counting beads. Results are expressed as the mean of the percentage of annexin V⁺/DAPI⁺ (±SEM) and the mean of the absolute number of viable cells (±SEM) of 2 independent experiments. *P ≤ .05.