IκBαSR inhibits in vitro B-lymphoid transformation by BCR-ABL1. (A) Ba/F3 parental cells or cells transduced with retrovirus expressing BCR-ABL1/GFP or BCR-ABL1/IκBαSR (4 × 10⁴) were plated in triplicate in the absence of IL-3, and viable cells determined by trypan blue staining. The difference in cell number between BCR-ABL1/GFP–expressing and BCR-ABL1/IκBαSR–expressing Ba/F3 at 96 hours was significant (P < .0001, Student t test). (B) Immunoblot of extracts from the cell lines in panel A, demonstrating expression of IκBαSR. (C) Representative confocal photomicrographs of nuclear RelA expression in Ba/F3 parental cells unstimulated or treated with TNFα (20 ng/mL) for 15 minutes, and Ba/F3 cells expressing BCR-ABL1/GFP or BCR-ABL1/IκBαSR. Cells were stained with antibody against RelA (red) and counterstained with Hoechst dye (blue). Scale bars = 10 μm. (D) Quantification of nuclear RelA fluorescence intensity per cell in the populations shown in panel B by confocal immunofluorescence microscopy. Data are presented as MFI of nuclear RelA staining relative to cells expressing BCR-ABL1/GFP (error bars indicate SE). The differences between unstimulated and TNFα-stimulated parental Ba/F3 cells (*), and between BCR-ABL1/GFP- and BCR-ABL1/IκBαSR-expressing Ba/F3 cells (**), were significant (P = .0003 and P = .0074, respectively, Student t test). (E) Transformation of primary B-lymphoid progenitors in vitro. BM was transduced with BCR-ABL1/GFP or BCR-ABL1/IκBαSR retrovirus and plated in triplicate on stroma at decreasing numbers of cells per well, as indicated by the colored lines. Positive wells were scored vs time when the viable nonadherent cell number reached 10⁶ per well. (F) Proliferation of primary B-lymphoid progenitors transformed by BCR-ABL1/GFP or BCR-ABL1/IκBαSR. Cells (4 × 10⁴) were seeded at day 0. Viable cells were determined by colorimetric assay for reduction of dimethylthiazol diphenyltetrazolium. The difference in viable cell number at 96 hours was significant (P < .0001, Student t test). (G) Representative confocal micrographs of nuclear RelA expression in B lymphoblasts expressing BCR-ABL1/GFP or BCR-ABL1/IκBαSR. Scale bars = 10 μm. (H) Nuclear RelA expression in the transformed cells from panel A was quantified by confocal microscopy, as described in “Materials and methods.” Data are presented as mean nuclear RelA fluorescence relative to cells expressing BCR-ABL1/GFP (error bars indicate SE). The average nuclear RelA content of BCR-ABL1/IκBαSR–transformed B-lymphoid cells was significantly decreased (*P = .0026, Student t test). MFI, mean of fluorescence intensity.