Fig. 7

The collaboration of RUNX1-R135T with ASXL1-R693X in the enhancement of HIF-1α and its target gene. a K562 cells were co-transfected with luciferase reporter plasmid of HRE promoter, pGL4.42[Luc-2p/HRE], pEGFP with EV, RUNX1-WT, and RUNX1-R135T plasmids; 32D cells with EV, ASXL1-R693X, RUNX1-R135T, and ASXL1-R693X + RUNX1-R135T. Forty-eight hours later, cells were incubated with 100 μM CoCl₂ for an additional 24 h. Increased folds of the relative HRE-Luc activities, which were normalized by GFP fluorescent intensity, were calculated against EV control (upper), and relative inhibitor of DNA binding 1 (ID1) mRNA levels was determined by real-time quantitative RT–PCR (lower). b Chromatin immunoprecipitation (ChIP) analyses were performed using the HIF1-α antibody in U937 cells overexpressed with ASXL1-R693X, RUNX1-R135T, ASXL1-R693X + RUNX1-R135T, and EV. ID1 expression was measured using real-time qPCR, and ChIP-qPCR data is displayed as enrichment relative to the input. All signals from transformed-U937 cells were standardized relative to the signal from control U937 cells, which were set to 1.0. Error bars represented mean ± SD based on two independent experiments. c, d Transformed K562 (c) and U937 cells (d) were cultured with 30 μM chrysin or no chrysin for 2 h. CoCl₂ (100 μM) was added, and the cells were incubated for another 12 h. The cells were harvested, lysed, and immunoblotted with indicated antibodies. e, f K562 cells were stably transduced with RUNX1-WT, RUNX1-R135T mutant, control (e), and U937 cells with ASXL1-R693X, RUNX1-R135T, and ASXL1-R693X + RUNX1-R135T including control (f). Cells were incubated in the presence of 30 μM HIF-1α inhibitor, chrysin, for 72 h, and viable cells were counted. Data represented here are the mean ± SD of duplicate cultures and experiments repeated three times. *P < .05, **P < .01, ***P < .001, compared with the control or as indicated in figures