Figure 1

Effects of ATRA and hypoxic signals on EPO production by hiPSC-EPO cells. (A) Semiquantitative RT-PCR analysis of the mRNA expression of RARs and RXRs by hiPSC-EPO cells. HepG2 cells, human fetal liver tissues and human skeletal muscle tissues were used as positive controls. Cropped gels are displayed. (B–E) Effects of ATRA treatment on EPO mRNA expression (B–D) and protein secretion (E) by hiPSC-EPO cells under normoxia (21% oxygen; B,E light gray), hypoxia (5% oxygen; C,E, dark gray) and normoxic conditions combined with PHD inhibitor treatment (10 μM FG4592; D,E, black), as analyzed by qRT-PCR and ELISA, respectively. Note that the analyses in (B–D) were performed independently. (F) Concentration-dependent effects of FG4592 on EPO protein secretion by hiPSC-EPO cells treated with 10 μM ATRA under normoxic conditions. (G) Effects of ATRA combined with several PHD inhibitors (100 μM molidustat, daprodustat and DFO, and 1 mM DMOG) on EPO protein secretion by hiPSC-EPO cells under normoxic conditions. (H,I) Effects of adding various concentrations of an RARα antagonist, AGN193109, to the ATRA treatment on EPO mRNA expression (H) and protein secretion (I) by hiPSC-EPO cells under hypoxic conditions. The data from four (n = 4 for B—E, H and n = 6 for I) or three independent experiments (n = 3 for F, G) are represented as the means ± SEM in (B–I). Statistical analysis was performed using one-way ANOVA with Dunnett’s multiple comparison test in (B–F,H,I) and Student’s t test in (G). ^#p < 0.05 versus the samples treated with DMSO under hypoxic conditions in (C,E) and those treated with ATRA but without AGN193109 under hypoxic conditions in (H,I). *p < 0.05 versus the samples treated with PHD inhibitors but without ATRA under normoxic conditions in (D,E,G). ^☨p < 0.05 versus the samples treated with DMSO under normoxic conditions in (E,F).