Journal: Leukemia

Article Title: BCR::ABL1 tyrosine kinase inhibitors induce ribosome collisions to activate ZAK-dependent ribotoxic stress and apoptosis in chronic myeloid leukemia

doi: 10.1038/s41375-026-02916-3

Figure Lengend Snippet: A Depletion of ZAK or a subset of RQC factors reduces cell proliferation in CML cell lines (K562 and KU812) but not in the non-CML cell line (Jurkat). Cells were split into 60-mm culture dishes 48 h after siRNA transfection and daily counted. Data represent mean ± SEM ( n = 3–6). n.s., not significant; ** P < 0.01, *** P < 0.001 to control siRNA, as determined by 1-way ANOVA, Dunnett’s multiple comparisons test. B , C ZAK depletion attenuates AKT-mTOR signaling in K562 cells. Total cell lysates were prepared 48 h after siRNA transfection and immunoblotted with the indicated antibodies. The relative abundance of phosphorylated proteins was calculated by normalizing the ratio of phospho-specific to total signals to that in control siRNA (set as 1). Data represent mean ± SEM ( n = 3). n.s., not sig n ificant; * P < 0.05, ** P < 0.01, *** P < 0.001 as determined by 1-way ANOVA, Dunnett’s multiple comparisons test. D, E ZAK overexpression upregulates AKT-mTOR signaling in a ZAK kinase activity-dependent manner. ZAK -deleted K562 cells ( ZAK KO ) were transfected with expression vectors for wild-type (WT), kinase-dead (K45A), or constitutively active ZAK (F368C). Total cell lysates were prepared 48 h after transfection and immunoblotted with the indicated antibodies. The relative abundance of phosphorylated proteins was calculated by normalizing the ratio of phospho-specific to total signals to that in the vector control. Data represent mean ± SEM ( n = 3). n.s., not significant; ** P < 0.01, *** P < 0.001 as determined by 1-way ANOVA, Tukey’s multiple comparisons test. F ZAK deletion impairs cell proliferation in K562 cells. Control (K562) and ZAK KO cells were split into 60-mm culture dishes and daily counted. Data represent mean ± SEM ( n = 4). *** P < 0.001 to control on the same day, as determined by 2-way ANOVA, Tukey’s multiple comparisons test. G ZAK sustains K562 cell proliferation in an AKT activity-dependent manner. Control (K562) and ZAK KO cells were counted 48 h after treatment with DMSO (vehicle control), SC79 (1 µM, AKT activator), or MK-2206 (1 µM, AKT inhibitor). Two-way ANOVA detected significant interaction effects of ZAK deletion with SC79 ( P = 0.0156) or MK-2206 ( P = 0.0003) on cell proliferation. Data represent mean ± SEM ( n = 7). n.s., not significant; *** P < 0.001 as determined by Tukey’s multiple compariso n s test.

Article Snippet: CCRF-CEM, MOLT-4, and Jurkat cell lines were purchased from the Korean Cell Line Bank.

Techniques: Transfection, Control, Over Expression, Activity Assay, Expressing, Plasmid Preparation