Fig. 1: Analysis of IKZF1 and IKZF3 expression.

Thirteen B-cell leukemia cell lines; 7 Ph-positive consisting of KOPN30bi (Ph1), KOPN55bi (Ph2), KOPN57bi (Ph3), KOPN66bi (Ph4), KOPN72bi (Ph5), KOPN83bi (Ph6), and KCB1 (Ph7) and 6 Ph-negative consisting of 2 TCF3-PBX1 positive, 3 ETV6-RUNX1 positive, and MLL-ENL positive, were used for analysis. A RT-PCR analysis of IKZF1 isoforms in B-cell leukemia cell lines. Primer sequences for RT-PCR analysis of IKZF1 were as follows: forward primer 5′-AAAGCGCGACGCACAAATCC-3′, reverse primer 5′-ATGGCGTTGTTGATGGCTTGGTC-3′⁴⁵. B Western blot analysis of IKZF1 expression before and after LEN treatment. B-cell leukemia cell lines were cultured for 24 h in the presence or absence of 10 µM LEN (Cayman Chemical, Ann Arbor, MI), and IKZF1 expression was examined on western blot. Anti-IKZF1 antibody was obtained from R&D Systems (Minneapolis, MN). C RT-PCR analysis of IKZF3 isoforms in B-cell leukemia cell lines. Primer sequences for RT-PCR analysis of IKZF3 were as follows: forward primer 5′-ATGGAAGATATACAAACAAATGCGGA-3′), reverse primer 5′-AGAGAGGCCTGTGTGAGAAGGCAC-3′⁴⁶. D Western blot analysis of IKZF3 expression before and after LEN treatment. B-cell leukemia cell lines were cultured for 24 h in the presence or absence of 10 µM LEN, and IKZF3 expression was examined on western blot. Anti-IKZF3 antibody was obtained from Proteintech (Rosemont, IL). E Comparison of decrease in IKZF1 and IKZF3 expression after LEN treatment in B-cell leukemia cell lines. In B and D, areas of IKZF1 (excluding Ik6) and IKZF3 were quantified by densitometry, and %decrease after LEN treatment was calculated as {1−[(area of LEN+)/(area of LEN−)]} × 100. The results were shown by box-whisker plots. Comparison was done by Mann–Whitney U test. Left panel: the %decrease in IKZF1 expression in Ph+ group A cell lines (n = 4, median 90.4, range 78.4–99.9) was significantly (p < 0.01) greater than that in non-Ph cell lines (n = 6, median 49.5, range 32.0–65.0). Right panel: the %decrease in IKZF3 expression in Ph+ cell lines (n = 6, median 85.0, range 58.7–89.7) was significantly (p < 0.01) higher than that in non-Ph cell lines (n = 6, median 50.2, range 36.3–67.5). F Time-course of IKZF1 and IKZF3 isoforms expression after LEN treatment. KOPN57bi cells were cultured with 10 µM LEN for 1, 3, 6, and 24 h, and changes in IKZF1 and IKZF3 expression was examined on western blot. G Effect of neddylation activating enzyme inhibitor MLN4924 on IKZF1 and IKZF3 isoforms expression after LEN treatment. KOPN57bi cells were cultured for 24 h in the presence or absence of 10 µM LEN with or without 30 min-pretreatment with 0.25 µM MLN4924 (ChemScene, Monmouth Junction, NJ), and changes in expression of IKZF1 and IKZF3 were examined on western blot.