Fig. 3

SUMOylation was required for sPRDM16 in the progression of acute myeloid leukemia. a Exogenous protein expression of sPRDM16 or K568R in THP-1 cell line. The original whole-cell lysates (WCL) were analyzed by immunoblotting (IB) with anti-FLAG or anti-actin antibodies. b THP-1 cells stably transfected with either vector, sPRDM16 or sPRDM16-K568R were seeded in 1 ml of medium containing 10 % FBS with 0.35 % soft agar at 1 × 10³ cells per well and layered onto the base soft agar medium. Photographs were taken after 12 days. Images are representative of three independent experiments. c The number of colonies of colony formation assay (A) was scored after 12 days. The colonies were visualized by staining with 0.5 % crystal violet. The experiments were analyzed in triplicate and colonies larger than 50 μm in diameter were counted under microscope. Columns, mean of triplicate experiments; bars, S.D. (* * P < 0.01). d Logarithmically growing THP-1 cells stably transfected with either sPRDM16 or sPRDM16-K568R vector, were exposed to 3 nM PMA for 24 h. The percentage of cells expressing the monocytic maturation marker CD11b was determined by flow cytometry. The dot plot results are representative of three independent experiments. e The results of flow cytometry described in Fig. 3C were represented as a histogram. Values represent the mean ± S.D. for experiments performed in triplicate. *P < 0.01 represents a significant difference from the cells treated with PMA (n = 3); columns, mean of triplicate experiments; bars, s.d. f Logarithmically growing THP-1 cells were treated with 3 nM PMA for 24 h. The percentages of adherence cells on the surface of plastic dish were counted. Values represent the mean ± S.D. for experiments performed in triplicate. *P < 0.01 represents a significant difference from the cells treated with PMA (n = 3). Columns, mean of triplicate experiments; bars, S.D. g sPRDM16 SUMOylation in THP-1 cells stably expressing sPRDM16-WT was decreased after incubation with PMA. SUMO1-conjugated proteins in THP-1 cells in the presence or absence of PMA for 24 h were immunoprecipitated with anti-FLAG M2 agarose beads. SUMO1-sPRDM16 proteins were blotted with anti-FLAG and anti-SUMO1. Cell lysate was immunoblotted with anti-FLAG and anti-FLAG antibody