Fig. 4

Phd2 bridges oxygen sensing mechanisms with Fgf23 production. A Phd2-KO cell line was generated in undifferentiated MPC2 cells using CRISPR/Cas9 technology and cells were differentiated for 2 or 3 weeks in osteogenic conditions. a Immunoblot of the Phd isoforms and HIF1α protein expression in 2- and 3-week differentiated WT and Phd2-KO cells. b Fgf23 expression in 3-week differentiated WT and Phd2-KO MPC2 cells (*P < 0.05 vs. WT cells). c Alizarin red stain to visualize mineral deposition in 3-week differentiated WT and Phd2-KO MPC2 cells. d Fgf23 gene expression and (D, inset) HIF1α protein expression in 3-week differentiated WT MPC2 cells treated with the HIF-PHI FG-4592 (Roxadustat) alone or with FG-4592 and 1 μmol·L⁻¹ or 5 μmol·L⁻¹ of the HIF1α inhibitor BAY 87-2243 for 48 h (**P < 0.01 versus vehicle; ^##P < 0.01 versus FG treatment alone). e Fgf23 expression and (inset) HIF1α protein expression in 3-week differentiated Phd2-KO cells treated with vehicle (DMSO) or with 1 μmol·L⁻¹ or 5 μmol·L⁻¹ of the HIF1α inhibitor BAY 87-2243 for 48 h (*P < 0.05 vs. veh). f Fgf23 expression in IDG-SW3 cells transduced with shRNA against HIF1α or HIF2α in vehicle or IOX2-treated cells (***P < 0.001 vs. Scr control, same treatment, ^###P < 0.001 vs. vehicle, same shRNA, ^^^P < 0.001 shHIF1 vs. shHIF2). g Tfrc, (g, inset) HIF1α protein, h Fgf23, i Phd1, and j Phd3 gene expression in 3-week differentiated WT (solid bars) and Phd2-KO (dashed bars) MPC2 cells treated for 24 h with 20 μmol·L⁻¹ or 50 μmol·L⁻¹ of the HIF-PHI FG (FG-4592; Roxadustat) or BAY (BAY85-3934; Molidustat) (*P < 0.05, **P < 0.01, ***P < 0.001 vs. veh, same genotype; ^#P < 0.05, ^###P < 0.001 WT vs. Phd2-KO, same treatment)