Fig 2: DNA binding and pericentromeric targeting of AIOLOS L162R. (a) EMSA was performed using nuclear extracts from HEK293T cells transfected with Flag-tagged AIOLOS WT or the mutant L162R. EV indicates the empty vector control. Nuclear extracts were subjected to Western blotting to test AIOLOS expression. Densitometry was performed to quantify AIOLOS binding to the IKBS1 probe, and the values were normalized to AIOLOS protein levels. The bar graph shows the relative IKBS binding levels normalized to WT samples. Data are expressed as mean + SD (n = 3). WB, Western blot. (b) NIH3T3 cells were transfected with Flag-tagged AIOLOS WT or the mutant L162R expression vector. Cells were stained with an anti-AIOLOS antibody, followed by Alexa Fluor 488–conjugated secondary antibodies. Cells were visualized by using an EVOS fluorescence microscope (20× objective). The scale bars indicate 25 µm. Data shown are representative of three independent experiments.

Fig 3: SUMOylation inhibition decreased IRF4 level independent of CRBN-IKF1/3 regulation.A TAK-981 synergizes with Len at increasing apoptosis marker cleaved PARP and decreasing IRF4 and c-Myc levels in MM. Western blot showing global SUMOylation (SUMO-2,3), cleaved PAPR, IRF4, and c-Myc level of MM1S and MMR10R (left) and H929 and KMS11 (right) cell lines. B Western blot showing CRBN, Aiolos (IKZF3) and Ikaros (IKZF1) level of MM1S and MMR10R (left) and H929 and KMS11 (right) cell lines. MM1S, MMR10R and H929 cells were treated with TAK-981 (0.1 µM) or Len 2.5 µM or both for 48 h; KMS11 cells were treated with TAK-981 (1 µM) or Len (25 µM) or both for 48 h. GAPDH was used as loading control. Relative protein level was quantified using Image J, normalized to GAPDH, and labeled below each blotting band.

Fig 4: Compound-mediated IKFZ3 degradation in OPM-2 cells. (A) Immunoblot analysis of IKZF3 levels after treatment with compounds 7d, 5a, and 7f (100 μM) for 24 h, compared to DMSO (control) and lenalidomide (20 μM) (n = 3). Lenalidomide at 100 μM reduced IKFZ3 levels effectively by 100% (not shown). (B) Averaged IKZF3 levels from the three independent experiments, normalized against total protein loading control (Figure S2). The significance of the data was tested comparing IKZF3 levels in the presence of the compounds and DMSO (p > 0.05).

Fig 5: Generation of Ikzf3N159S mouse strain and nonlymphoid immune cells in mice. (a) Sanger sequencing of the surrounding region in the Ikzf3 N159S from mice with the indicated genotype. Upper panel shows the designed mutation for the Ikzf3N159S. (b) Table showing the number of 1-wk-old mice with the indicated genotype that were born by heterozygous-to-heterozygous crossing. (c) Immunoblot showing Aiolos protein expression in total thymocytes. Gapdh was used as a loading control. Data are representative of three independent experiments. (d) Total splenocytes were gated on the B220−TCRβ− population, and then analyzed dendritic cells (DC; CD11b+CD11c+), neutrophils (Neu; CD11b+CD11c− Ly6G+), NK cells (CD11b+CD11c−NK1.1+), and eosinophils (Eos; CD11b+CD11c−NK1.1−Siglec-F+) as shown in the figure. FSC, forward scatter. (e) Graphs show the frequency (top) and absolute numbers (bottom) of indicated populations from WT Ikzf3+/+ littermates (filled circle), Ikzf3+/N159S heterozygous mice (filled triangle), and Ikzf3N159S/N159S homozygous mice (open circle). Horizontal lines indicate the median values of at least three different mice (2–3 mo old) per group. Statistically significant differences (Student’s unpaired, two-tailed t test) between the groups are shown. *, P < 0.05; **, P < 0.01; ***, P < 0.001.