Low iron status alters VEGF and ERK signaling in MEPs. (A) Volcano plot of RNAseq analysis of sorted MEPs from Tmprss6^−/− and WT mice. Upregulated genes, including VEGF targets, are identified by orange dots. Downregulated genes, including ERK targets, are identified by blue dots. (B) Representative histograms of phospho-ERK (pERK) in GFP⁺ gated human MEP transduced with scrambled shRNA (purple line) or TFR2-directed shRNA (green line); immunoglobulin G (IgG) control (red dotted line). (C) Normalized fluorescence intensity of phospho-ERK in GFP⁺ transduced human MEPs (n = 4 per group). (D) Representative histograms of phospho-ERK in WT (purple line) and Tmprss6^−/− (green line on left graph) MEPs, WT MEPs treated with VEGF (50 ng/mL) for 30 minutes (green line on right graph), and IgG control (red dotted line). (E) Normalized MFI of phospho-ERK shown in panel D normalized to untreated WT MEPs (n = 3). (F) Representative histograms of phospho-ERK in untreated human MEPs (purple line), human MEPs treated with VEGF (50 ng/mL) for 30 minutes (green line), and IgG control (red dotted line). (G) Normalized MFI of phospho-ERK shown in panel F, normalized to untreated human MEPs (n = 3). (H) Colony counts by type per 100 MEPs plated in the presence of vehicle control (DMSO) or ERK inhibitor (PD98059) (n = 4). Data are presented as mean + SD. *P < .05, **P < .01, ***P < .001, and ****P = .0001 calculated by 2-tailed unpaired Student t test. FC, fold change.