The 3 cysteine residues are important for HMGB1 function in erythropoiesis. (A-D) HUDEP-2 cells were treated with WT or mutated (3S) HMGB1. (A) Cell growth measured at indicated days of differentiation. (B) Cell pellets demonstrating the extent of hemoglobinization at D5. (C) Annexin V staining of HUDEP-2 cells at indicated days of culture. (D) Representative Western blot of cleaved caspase-3 levels at D5. (E) Surface plasmon resonance analysis of EPO (purple line), vehicle (blue line), and 3S HMGB1 (red line) binding to the extracellular domain of EPO-R. (F) Surface plasmon resonance analysis of vehicle (blue line) and 3S HMGB1 (red line) binding to EPO. (G) HUDEP-2 cells were preincubated with WT or 3S HMGB1 for 1 hour followed by a 4-hour pulse of EPO. pSTAT5 levels measured by western blot. Data presented as mean ± SEM. One-way ANOVA with Tukey’s post hoc test: *P < .05, **P < .01.