Fig 1: PGC1ß knock-down results in deregulated hemoglobinization of erythroid progenitors. (A) Representative flow cytometry plots of bone marrow derived erythroid progenitors within GPA + (SCR). (B, C) RT-qPCR analysis of gene expression relative to GAPDH on day 18 of genes important for Oxidative phosphorylation (OXPHOS) in bone marrow derived (B) polychromatic erythroblasts and (C) orthochromatic erythroblasts respectively (n = SCR:4, Sh3:12, Sh5:6). (D) Representative flow cytometry plots of cord blood derived erythroid progenitors within GPA + (SCR). (E, F) RT-qPCR analysis of gene expression relative to GAPDH on day 16 of genes important for Oxidative phosphorylation (OXPHOS) in cord blood derived (E) polychromatic erythroblasts and (F) orthochromatic erythroblasts respectively (n = 4). (G) Mitochondria related protein levels were evaluated by western blot in sorted cord blood derived polychromatic erythroblasts from day 16 of culture (ALAS2 (65 kDa) specific bands appear at 120 kDa, likely due to protein dimers). Western blot quantification of (H) TOM20 relative to ß-Actin as a marker for mitochondrial mass in relation to cell number, and (I) OXPHOS related ATP5S and NDUFA1 relative to TOM20/ß-Actin to account for differences in mitochondrial mass. Expression depicted as normalized to control (SCR, n = 4). (J) Quantification of mitochondrial membrane potential of cord blood derived polychromatic erythroblasts measured on day 16 of differentiation using TMRE and flow cytometry (n = 4). (K) Quantification of ROS production by mitochondria in cord blood derived polychromatic erythroblasts measured on day 16 of differentiation using MitoSox and flow cytometry (n = 4). (L, M) RT-qPCR analysis of gene expression relative to GAPDH on day 18 of genes important for hemoglobin and heme synthesis of bone marrow derived polychromatic erythroblasts (left) and orthochromatic erythroblasts (right) respectively (n = SCR:4, Sh3:12, Sh5:6). (N) Representative images of DAB-Giemsa stained erythroblasts from d21 of culture at 40 × magnification and (O) quantification of hemoglobin content using ImageJ software (n = SCR:4, Sh3:12, Sh5:6). (P, Q) RT-qPCR analysis of gene expression relative to GAPDH on day 16 of genes important for hemoglobin and heme synthesis in cord blood derived polychromatic erythroblasts and orthochromatic erythroblasts respectively (n = 4). (R) Western blot quantification of heme related ALAS2 and FECH relative to ß-Actin and (S) TOM20/ß-Actin to account for differences in mitochondrial mass (n = 4). Expression depicted as normalized to control (SCR. Data is presented as mean ± SEM (*P = 0.05, **P = 0.01, ***P = 0.001, ****P = 0.0001).