Figure 2.

Small molecule screen identified histone lysine demethylase inhibitor IOX1 as a potential compound to downregulate α-globin expression. (A) A schematic of the work flow of the small molecule screen. Small molecules were added to the liquid culture medium on day 7 of erythroid cell differentiation (corresponding to the proerythroblast stage), and the cells were then incubated in a 5% CO₂ atmosphere at 37°C for 72 hours. Gene expression was analyzed using the Fluidigm high throughput qPCR system. (B) Representative heat map (one of 3 biological repeats) demonstrating fold differences of α- and β-globin mRNA levels in erythroid cells treated with small molecules. The expression levels were normalized to multiple housekeeping genes (RPL13A, RPL18, GAPDH and FTH1) and referenced to the vehicle (DMSO) control. Each row represents a single compound and each column represents one of three technical repeats performed for the α- and β-globin genes, each with colors ranging from dark red (downward expression) to blue (upward expression); HBA, α-globin; HBB, β-globin. (see Online Supplementary Figure S3 for full heat map). Four compounds that downregulate α-globin expression are marked using green rectangles. (C) α/β-globin mRNA ratios in erythroid cells (differentiated from cord blood CD34⁺ cells) treated with a dose range of IOX1 analyzed by qPCR. Error bars represent SD (n=3); *P<0.05, **P<0.01 relative to DMSO control. (D) Globin mRNA levels in erythroid cells (differentiated from cord blood CD34⁺ cells) treated with IOX1 (40μM) quantified by nCounter Digital Analyzer (NanoString Technologies). The NanoString count for each globin gene was normalized to the counts of multiple housekeeping genes (RPL13A, RPL18, GAPDH, PABPC1, CA2, FTH1, PAIP2 and LAPTM4A). Error bars represent SD (n=3); *P<0.05, **P<0.01 relative to DMSO control. DMSO: dimethyl sulfoxide; mRNA: messenger ribonucleic acid; RNA: ribonucleic acid.