Figure 4.

EVI1 and ATRA synergistically induce GDF15 protein in myeloid and in epithelial cells. (A) U937_vec and U937_EVI1 cells were treated with solvent or ATRA for 3 or 5 days, and the intracellular precursor and secreted mature forms of GDF15 were detected in whole cell extracts (day 3) and culture supernatants (day 5), respectively, by immunoblot analysis. Both forms are disulfide linked homodimers, and the corresponding 35 kDa and 15 kDa monomers are detected after reducing SDS-PAGE. (B) U937_vec and U937_EVI1 cells were treated with solvent or ATRA for 3 or 5 days, and mature GDF15 was detected in culture supernatants by ELISA. Results represent means + SEs from 3 independent experiments. Significance was calculated using Student's 2-tailed t-test (*, P < 0.05; **, P < 0.01; ***, P < 0.001). (C) The nontumorigenic prostate epithelial cell line BPH-1 and its transformed derivative, BPH-1 CAFTD03, were subjected to immunoblot analysis to detect endogenous EVI1. (D) BPH-1 and BPH-1 CAFTD03 cells were treated with 1 or 2 μM ATRA for the indicated times, and intracellular GDF15 levels were determined by immunoblot analysis.