Fig. 5

Collaborative mutations of ASXL1 and RUNX1 enhanced ID1 expression. a, b mRNA expression of Id1 was analyzed by RT-qPCR in the spleen (a, left, n = 3), liver (a, right, n = 3), and BMC (b, n = 2) from killed or moribund mice transplanted with control, ASXL1-R693X, RUNX1-R135T, and combined mutations of ASXL1 and RUNX1. Error bars represent the mean ± SD of two repetitions. c Immunoblot analyses of ID1 in the liver and spleen from BMT-mice. d Relative mRNA expression of ID1 in BM leukemic cells from patients harboring either ASXL1-MT (n = 8) or RUNX1-MT (n = 7) and coexisted RUNX1 and ASXL1 mutations (n = 5). Gene expression was measured using real-time qPCR and shown as a relative to normal bone marrow cells (average of 3). NC indicated normal controls. e Immunoblot analyses with indicated antibodies in transduced U937 cells (left), K562 cells (middle), and 32D cells (right); β-actin representing as an internal control. f Knocked down ID1 in the transduced U937 and K562 cells using two different shRNA and scrambled (shLuc), and silencing efficiency was checked by immunoblot analyses after 5 days. g Silenced cells were incubated for 72 h, and cell proliferation was measured by trypan blue exclusion method. Data represented here are the mean ± SD of triplicate cultures, and experiments repeated twice. P value showing in the figure calculated either compared with the control or as indicated in figures. The values (c) indicating relative signal density corresponding to actin expression