Fig. 3

RUNXs inhibit CRBN-dependent IKZF1 and IKZF3 degradation. a Immunoblot analysis of whole cell lysates from OPM-1 cells treated with 1 μM lenalidomide for the indicated durations of time. b Same as in (a) except that OPM-1 cells were treated with lenalidomide for the indicated time points. c Immunoblot analysis of whole cell lysates from OPM-1 cells treated with at the indicated concentrations of lenalidomide for 36 h. d HEK293T cells were transfected with FLAG-CRBN, HA-IKZF1, and un-tagged RUNX1. Where indicated, cells were treated with 2 μM lenalidomide for 6 h before harvesting. The cell extracts were subjected to anti-FLAG IP and the immunocomplexes were probed with antibodies to the indicated proteins. A low exposure (l.e.) and high exposure (h.e.) are shown for IKZF1. e Same as in d except that HEK293T cells were transfected with FLAG-IKZF1 and un-tagged RUNX1. f Chromatin immunoprecipitations of IKZF1 in OPM-1 cells coupled with qRT-PCR using primers for IRF4 promoter under the indicated conditions. Red bar shows the distance between the primers and transcription start site of IRF4 (n = 2 independent experiments). g Levels of IRF4 and MYC mRNA were analyzed by qRT-PCR under the indicated conditions (mean ± s.d., n = 2 independent measurements). h Cell counts of GFP/Cherry-sorted OPM-1 RUNX1^+/+ RUNX3^+/+ or RUNX1^−/−RUNX3^−/− cells grown in media containing DMSO or 0.1 μM lenalidomide (mean ± s.d., n = 3 independent experiments, two-way ANOVA, n.s., not significant, ****P ≤ 0.0001). Unless otherwise noted, immunoblots are representative of three independent experiments