HPK1-C increases AICD of B cells by the intrinsic cell death pathway. (A) Mouse splenic B cells were analyzed for AICD by incubation with 1 or 10 μg/mL of plate-bound anti-IgM antibodies for 18 hours in the presence (▩) or the absence (□) of 10 μg/mL of the CD95L-blocking antibody MFL3. Standard deviation is given for triplicate measurements. (B) Splenic B cells were analyzed for AICD by incubation with 1 μg/mL of plate-bound anti-IgM antibodies in presence or absence of the caspase-9–specific inhibitor z-LEHD-fmk (40 μM) for 18 hours. Standard deviation is given for triplicate measurements. (C) WEHI-231 B cells were activated by BCR cross linking using 5 or 10 μg/mL of plate-bound anti-IgM antibodies. Cells were lysed and presence of HPK1 and processing toward HPK1-C were analyzed by Western blotting. (D) Splenic B cells of HPK1-C transgenic (tg) mice or non-transgenic (non-tg) littermates were analyzed for AICD by incubation with 1 or 10 μg/mL of plate-bound anti-IgM antibodies for 18 hours. Standard deviation is given for triplicate measurements. The inset shows expression levels of HPK1 and HPK1-C as detected by Western blotting. (E) A GFP:HPK1-C fusion protein¹⁹  (HPK1-C) or GFP alone (vector) was expressed in WEHI 231 B cells by transient transfection and GFP-positive cells were analyzed for cell death upon incubation with 10 μg/mL of anti-IgM antibodies for 12 hours. Standard deviation is given for triplicate measurements.