DYRK1a-NIK axis mediates B-ALL cell survival. (A) Flow cytometric analysis of BAFFR expression in human B-ALL cells. The Burkitt lymphoma cell line DAUDI was used as a negative control. (B) Immunoblot analysis of the indicated proteins in the cytoplasmic or nuclear extracts of BALL1 cells transduced with either a control nonsilencing shRNA or two different DYRK1A shRNAs and stimulated with BAFF. (C) Viable cell quantification by flow cytometry, based on SYTOX blue cell staining, of control BALL1 cells (shControl), DYRK1A-knockdown (using DYRK1A shRNA #1) BALL1 cells (shDYRK1A), or DYRK1A-knockdown BALL1 cells and transduced with the NIK expression vector (shDYRK1A + NIK), cultured in the presence of BAFF or medium control. Data are mean ± SD values combined from the mean values of 3 independent experiments. (D) Flow cytometric analysis of viable and dead cells, based on SYTOX blue cell staining, in BALL1 cells or BALL1 cells transduced with a NIK expression vector (BALL1-NIK). The cells were cultured for 8 days in the presence of BAFF or medium control along with the DYRK1a inhibitor EHT1610 (10 µM) or DMSO. The summary graph is based on 3 replicate samples of the same experiment. (E) Immunoblot analysis of p100 and its processing product p52 in BALL1 or BALL1-NIK cells cultured for 8 days in the presence (+) or absence (–) of BAFF and EHT1610. Summary graph presents p52/p100 ratio based on densitometric quantification of immunoblot protein bands in 3 independent experiments. P values are determined by an unpaired, 2-tailed Student t test (D-E) or 2-way ANOVA with Bonferroni correction (C). *P < .05; **P < .01; ***P < .001.