FIG 4.

Effects of ASH2L depletion in U937 leukemic cells. (A) Knockdown of ASH2L in U937 cells. Human U937 leukemic cells were infected and selected as indicated in the legend to Fig. 1C. Equal amounts of cell extracts from mock knockdown (shRandom) and interfering-RNA cells (shUTX or shASH2L) were analyzed by Western blotting with the indicated antibodies. Knockdown in shUTX U937 cells is shown as a control. (B) Downregulation of ASH2L affects U937 differentiation after treatment with RA. shRandom or shASH2L U937 cells were treated with RA for 24 and 48 h or untreated, and cell differentiation was evaluated as percent CD11b-positive cells, as indicated for Fig. 1D. Error bars represent standard deviations from the mean for triplicate experiments. Differentiation in shUTX U937 cells is shown for comparison. (C) Knockdown of ASH2L affects RAR target gene expression. Total RNA from shRandom or shASH2L U937 cells that were untreated (−RA) or treated with RA (+RA) was prepared. mRNA levels of the genes indicated were analyzed relative to a PUM1 control by RT-qPCR. Results are presented as the means and SEM from three independent experiments. (D) Downregulation of ASH2L did not impair trimethyl H3K27 (3meH3K27) levels at RAR target genes. shRandom or shASH2L U937 cells that were untreated (−RA) or treated with RA (+RA) were subjected to ChIP analysis using the indicated antibodies. IgG was included as a control. Error bars indicate standard deviations obtained from three independent experiments.