Fig. 1: Ikaros dissociates from metaphase chromosomes but can be retained following treatment with specific kinase inhibitors.
a Ikaros staining in fixed, asynchronous mouse preB cells, showing representative interphase and mitotic stages. Scale bar = 5 µm; ≥ 12 cells imaged per stage (> 50 metaphase) across four independent experiments. b CRISPR/Cas9-mediated knock-in (KI) of mNeonGreen at endogenous Ikzf1 in mouse preB cells (illustrated top). Primers (blue arrows) were used to confirm KI by PCR (middle, two clones per targeting). Sequencing revealed clones 1.1 and 1.2 are heterozygous (Ikzf1mNG/–, apparent WT alleles harbour frameshift mutations); clones 2.1 and 2.2 are homozygous (Ikzf1mNG/mNG). Immunoblotting (bottom) detects Ikaros-mNeonGreen (anti-Ikaros(C-terminal), 1:5000); anti-H3 = loading control. c Live-cell imaging of Ikaros-mNeonGreen in asynchronous Ikzf1mNG/mNG mouse preB cells (clone 2.1) cultured with SiR-DNA, showing representative interphase and mitotic stages. Scale bar = 5 µm; ≥ 11 cells imaged per stage ( > 50 metaphase) across four independent experiments. d Live-cell imaging of mitotic Ikzf1mNG/mNG mouse preB cells (clone 2.1) from asynchronous cultures treated 10 min with DMSO or kinase inhibitors (K252a 1 µM, others 10 µM; Alp = alsterpaullone). Representative of at least two independent experiments (five for K252a and OTS514); scale bar = 5 µm. Chromosomal versus cytoplasmic mNeonGreen intensity is quantified (illustrated in Supplementary Fig. 1e) for a representative replicate of each treatment (n = 20, 27, 25, 18, 28, 26 cells/treatment; boxplots show median, interquartile range, Tukey whiskers (log2 scale); ****padj < 0.0001, two-tailed Dunnett’s test). e Ikaros staining in mitotic preB cells (fixed asynchronous cultures) following 10 min DMSO/OTS514 (10 µM) treatment. Representative of three independent experiments. All (63/63) OTS514-treated metaphase cells showed bright centromeric staining, versus 0/66 for DMSO. Scale bar = 5 µm. f Ikaros staining in mitotic VL3-3M2 mouse T cells (fixed asynchronous cultures) following 10 min DMSO/OTS514 (10 µM) treatment. Representative of three independent experiments; 79% of OTS514-treated metaphase cells showed centromeric staining (n = 80 cells; 34 = bright, 29 = mid, 17 = low/none), versus 17% of DMSO-treated cells (n = 75 cells; 0 = bright, 13 = mid, 62 = low/none). Scale bar = 5 µm. g Ikaros staining in mitotic J774A.1 mouse macrophages (fixed asynchronous cultures) after 10 min DMSO/OTS514 (10 µM) treatment. Representative of two independent experiments. All (42/42) OTS514-treated metaphase cells showed bright centromeric staining, versus 0/46 for DMSO. Scale bar = 5 µm. Source data for Fig. 1b, d are provided as a Source Data file.
