Figure 1
Figure 1. Pre-BCR signaling and Ikaros up-regulate BCL6 in normal and leukemic pre-B cells. Ighm−/− pro-B cells and Blnk−/− pre-BII cells were transformed with BCR-ABL1 and pre-BCR signaling was reconstituted by retroviral expression of BLNK-GFP, CD8/μ-chain or GFP and CD8 empty vectors. BCL6 mRNA levels were measured by quantitative RT-PCR in (A; n = 2) and protein levels were determined by Western blot in (B; n = 2) using β-actin as loading control. IL-7–dependent pre-B cells from Rag2−/− tTA/μ-chain-transgenic mice2 were cultured in the presence of tetracycline, removal of which induced activation of transgenic μ-chain expression under endogenous transcriptional control elements. Inducible differentiation was verified by flow cytometry (supplemental Figure 2) and BCL6 and MYC protein levels were measured by Western blot (C; n = 2). Normal IL-7–dependent pre-B cells were transduced with a constitutively active (CA) mutant of FoxO1 or an empty vector (transduction efficiency shown in supplemental Figure 1). After inducible activation with 4-hydroxy-tamoxifen (4-OHT), cells were subjected to Western blot analysis for BCL6 and β-actin as loading control (D; n = 2). Likewise, normal IL-7–dependent pre-B cells from Ptenfl/fl mice (supplemental Table 4) were transduced with 4-OHT–inducible Cre-ERT2 or an ERT2 empty vector control and subjected to antibiotic selection. Cre-mediated deletion of Pten and BCL6-up-regulation on IL-7 withdrawal was studied by Western blot using β-actin as loading control (E; n = 2). Pre-B ALL cells from Bcl6+/+ bone marrow were transduced with retroviral expression vectors for BCL6-GFP or a GFP empty vector control. The relative change of the percentage of GFP+ cells over time was measured by flow cytometry (F; n = 2). The cell-cycle profile of transduced cells was measured by BrdU incorporation and flow cytometry (G; n = 2) with statistical analysis (H). Pre-B ALL cells transduced with BCL6-GFP or GFP alone were sorted for GFP and transplanted into sublethally irradiated NOD/SCID mice via tail vein injection (3 mice per group). Spleens of recipient mice and their weight (right) are shown (I).

Pre-BCR signaling and Ikaros up-regulate BCL6 in normal and leukemic pre-B cells.Ighm−/− pro-B cells and Blnk−/− pre-BII cells were transformed with BCR-ABL1 and pre-BCR signaling was reconstituted by retroviral expression of BLNK-GFP, CD8/μ-chain or GFP and CD8 empty vectors. BCL6 mRNA levels were measured by quantitative RT-PCR in (A; n = 2) and protein levels were determined by Western blot in (B; n = 2) using β-actin as loading control. IL-7–dependent pre-B cells from Rag2−/− tTA/μ-chain-transgenic mice2  were cultured in the presence of tetracycline, removal of which induced activation of transgenic μ-chain expression under endogenous transcriptional control elements. Inducible differentiation was verified by flow cytometry (supplemental Figure 2) and BCL6 and MYC protein levels were measured by Western blot (C; n = 2). Normal IL-7–dependent pre-B cells were transduced with a constitutively active (CA) mutant of FoxO1 or an empty vector (transduction efficiency shown in supplemental Figure 1). After inducible activation with 4-hydroxy-tamoxifen (4-OHT), cells were subjected to Western blot analysis for BCL6 and β-actin as loading control (D; n = 2). Likewise, normal IL-7–dependent pre-B cells from Ptenfl/fl mice (supplemental Table 4) were transduced with 4-OHT–inducible Cre-ERT2 or an ERT2 empty vector control and subjected to antibiotic selection. Cre-mediated deletion of Pten and BCL6-up-regulation on IL-7 withdrawal was studied by Western blot using β-actin as loading control (E; n = 2). Pre-B ALL cells from Bcl6+/+ bone marrow were transduced with retroviral expression vectors for BCL6-GFP or a GFP empty vector control. The relative change of the percentage of GFP+ cells over time was measured by flow cytometry (F; n = 2). The cell-cycle profile of transduced cells was measured by BrdU incorporation and flow cytometry (G; n = 2) with statistical analysis (H). Pre-B ALL cells transduced with BCL6-GFP or GFP alone were sorted for GFP and transplanted into sublethally irradiated NOD/SCID mice via tail vein injection (3 mice per group). Spleens of recipient mice and their weight (right) are shown (I).

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