Fig. 3.

Overexpression of anti-apoptotic BCL2 blocks cell death by CHK1i in vitro a Pro B cells (NK1.1⁻ B220⁺ CD19⁺ AA4.1⁺ μHC⁻ CD25⁻ ckit⁺) were isolated from wild-type and Chk1 ^+/− C57Bl/6N mice and expanded for four days in IL-7 containing medium (n = 3/genotype). At day 4 cells were counted to assess cell expansion (left) and treated for additional 24 h with graded doses of the CHK1 inhibitor PF-477736 (right). Survival was assessed using AnnexinV-staining and flow cytometry. b FACS-sorted wild-type (WT) and BCL2 transgenic (Vav-BCL2 ⁺) bone marrow derived B220⁺ IgM⁻ pro/pre B cells were treated for 24 h and 48 h with PF-477736 or solvent control (DMSO). Cells were processed for survival analysis using a flow cytometer and AnnexinV/7AAD staining. c, d Total splenocytes were cultured for the indicated time in the presence of PF-477736 or solvent control (DMSO) and processed for survival analysis using B220/TCRβ/AnnexinV-staining and flow cytometry. c Quantification of B220⁺ AnnexinV⁻ cells. d Quantification of TCRβ⁺ AnnexinV⁻ cells. Bars in b–d represent means of n = 3 animals/genotype ± S.E.M. *p < 0.05, **p < 0.01, ***p < 0.001 using unpaired Student´s t test