HMGB1 leads to death of erythroid precursors by inhibiting the EPO and mTOR signaling pathways. (A) Quantification of Annexin V staining in specific erythroid precursor populations by flow cytometry at D11 of differentiation. Data represented as mean ± SEM. Unpaired, 2-tailed Student t test: *P < .05; ns, not significant. (B) Apoptosis array performed on erythroid cells at D11. Cleaved casapse-3 is boxed. (C-D) Representative western blot of cleaved caspase-3 levels at D11 with accompanying quantification. Data represented as mean ± SEM. Unpaired, 2-tailed Student t test: *P < .05. (E) Bubble plot representation of EPO downstream effectors activation by phosphoflow cytometry. Human erythroid cells at D7 of differentiation were preincubated with HMGB1 and then pulsed with EPO for indicated times. (F) pJAK and pSTAT5 levels in HUDEP-2 cells preincubated with HMGB1 for 1 hour followed by a 4-hour pulse with EPO (i). Quantification of pJAK2 and pSTAT5 levels normalized to GAPDH (panels ii and iii). Data represented as mean ± SEM. One-way ANOVA with Tukey’s post hoc test: *P < .05; ns, not significant. (G) pSTAT5 and pS6 levels in CD34⁺ cells at D7 preincubated with HMGB1 for 1 hour followed by 30-minute pulse with 3 or 30 U/mL of EPO (i). Quantification of pSTAT5 and pS6 levels normalized to STAT5 and S6 (ii). (H) pSTAT5 and pS6 levels in CD34⁺ cells at D7 preincubated with 10 μg/mL HMGB1, 10 nM IL-1, 100 ng/mL IL-6, or 20 ng/mL TNF for 1 hour followed by 30-minute pulse with EPO (i). Quantification of pSTAT5 and pS6 levels normalized to STAT5 or S6 (ii). Data represented as mean ± SEM. One-way ANOVA with Tukey’s post hoc test: *P < .05; **P < .01; ***P < .001.