Fig. 2

RUNX1-R135T cooperatively affected ASXL1 mutant K562 cells. a Endogenous expression of ASXL1 and RUNX1 in different leukemia cell lines including the ASXL1-mutated K562 cell line. b Stably transduced RUNX1-WT/MT in K562 cells and showing BCR-ABL, ABL, EZH2, and histone-modifying protein expression in transformed cells by immunoblot analysis. c, d K562 cells were stably transduced with WT- and RUNX1-R135T mutants, then cell proliferation was checked by trypan blue exclusion method (c) and colony formation ability was assayed in methylcellulose containing complete RPMI medium (d). After 10 days, colonies were photographed and counted (original magnification × 100). e Cell morphology analyzed by modified Wright–Giemsa-stained smears after treatment with 40 nM PMA in EV, RUNX1-WT, and RUNX1-R135T-transduced K562 cells for 96 h, magnification × 400 (left, upper panel), and digital images were acquired using Olympus (model no. U-TV0.5XC-3) microscopes equipped with a digital camera. Flow cytometry analyses of megakaryocytic marker (CD61) after the treatment of PMA in transduced K562 cells; a representative experiment is shown (left, lower panel); the percentage of CD61 expressing K562-positive cells are shown (right). f Analysis of mRNA levels of HOXA5, HOXA7, HOXA9, and HOXA10 by RT-qPCR after overexpression of RUNX1-R135T and EV in K562 cells. Error bars represent mean ± SD based on three independent experiments. *P < .05, **P < .01, ***P < .001, either compared with the control or as indicated in figures; n.s., not significant