HMGB1 can interact with the extracellular domain of the erythropoietin receptor and EPO. (A) HUDEP-2 cells preincubated with HMGB1 ± increasing concentrations of NSC87877 followed by a 4-hour pulse of EPO. pSTAT5 levels measured by western blot. (B) HUDEP-2 cells were preincubated with HMGB1 ± sodium orthovanadate followed by a 4-hour pulse of EPO. pSTAT5 levels measured by western blot. (C) Transcriptome analysis of EPO-R and CD117 expression on sorted stages during human erythropoiesis adapted from Yan et al.¹⁸ (D) SPR analysis of EPO (purple line), vehicle (blue line), and HMGB1 (green line) binding to the extracellular domain of EPO-R. (E) SPR analysis of vehicle (blue line) and HMGB1 (green line) binding to EPO. (F) Quantification of HMGB1 binding to EPO in the presence or absence of EPO-R in the analyte as assessed by SPR. Data represented as mean ± SEM. Unpaired, 2-tailed Student t test: **P < .01. (G) HUDEP-2 cells were either preincubated with HMGB1 followed by a pulse of EPO or cotreated with HMGB1 and EPO. pSTAT5 levels following 1-hour pulse of EPO. (H) UT7-Epo cells were preincubated with increasing concentrations of HMGB1 followed by a 30-minute pulse of 0.03, 0.3, or 3 IU/mL EPO. pSTAT5 and pS6 levels measured by western blot. Quantification of pSTAT5 and pS6 levels normalized to STAT5 or S6. Data represented as mean ± SEM. One-way ANOVA with Tukey’s post hoc test: *P < .05; **P < .01; ****P < .0001.