Fig. 3: The essential role of BGLT3 in induction of γ-globin by RK-701.

a ChIP-seq traces for H3K9me2 on the β-globin locus in HUDEP-2 cells after 4-day treatment with 1 μM RK-701 or DMSO. A representative image of two independent experiments is shown. b Diagram of the human β-globin locus. Positions of amplicons used for ChIP-qPCR are indicated. c ChIP-qPCR analyses using antibodies against IgG or H3K9me2 in HUDEP-2 cells treated with 1 μM RK-701 or DMSO for 4 days. Data are mean ± SD from three independent experiments. d A volcano plot illustrating changes in gene expression induced by RK-701. RNA-seq analysis was performed in HUDEP-2 4 days after treatment with 1 μM RK-701 or DMSO. The plot represents statistical significance versus fold change in gene expression between the two conditions. Results from three biological replicates are shown. Red, gray, or blue dots indicate differentially expressed genes (DEG) for upregulated, not significant or downregulated. Some of the data points that are out of scale indicate the value of inf (infinite) in Supplementary Table 6. e Relative expression of BGLT3 RNA in HUDEP-2 cells treated with RK-701 or RK-0133114 for 4 days. Data are mean ± SD from three independent experiments. f Relative expression of BGLT3 RNA in CD34⁺ cells treated with RK-701 or hydroxyurea on day 7 or day 9 of erythroid differentiation. Data are mean ± SD from three independent experiments. g–i Effects of G9a knockdown on γ-globin and BGLT3 gene expression in HUDEP-2 cells. G9a knockdown efficiency was evaluated by western blotting (g). A representative image of two independent experiments is shown. Relative expression of β- and γ-globin (h) and BGTL3 (i) in HUDEP-2 cells was analyzed by RT-qPCR. Data are mean ± SD from three independent experiments. j, k Effects of BGLT3 knockdown on the RK-701–induced expression of BGLT3 or γ-globin. Relative expression of BGLT3 (j) or γ-globin (k) in BGLT3-knockdown HUDEP-2 cells treated with 1 μM RK-701 for 4 days was analyzed by RT-qPCR. Data are mean ± SD from three independent experiments. P-value was calculated by the one-way ANOVA with Tukey’s post hoc test.