Fig. 6: TLR8 impairs erythropoiesis by interfering with the ANXA2-mediated plasma membrane localization and activation of STAT5.

a The percentage of CD71⁺CD235a⁺ cells and cell number from days 4 to 8 of erythroid differentiation (CB-derived CD34⁺ cells). b Immunoblotting of STAT5 phosphorylation in cells treated with VTX2337 or CUCPT8m from days 0 to 11 during erythroid differentiation and the quantitation of relative p-STAT5 levels (n = 3 biologically independent experiments). c Immunoblotting showing the effects of increasing VTX2337 dosage on STAT5 phosphorylation in HuDEP2 cells and the quantitation of relative p-STAT5 levels (n = 3 biologically independent experiments). d Immunoblotting (left) showing the effects of increasing duration of 5 μM VTX2337 on p-STAT5 phosphorylation in HuDEP2 cells and the quantitation of relative p-STAT5 levels (n = 3 biologically independent experiments). e Immunoblotting of the indicated proteins of primary erythroid cells on day 6 of erythroid differentiation treated with 10 μM VTX2337/CUCPT8m (n = 3 biologically independent experiments) and f the quantitation (n = 3 biologically independent experiments). g Co-immunoprecipitation of ANXA2/STAT5 from HuDEP2 cells treated with 10 μM VTX2337 for 2 h (n = 3 biologically independent experiments). h Immunofluorescence of STAT5 in HuDEP2 cells treated with or without VTX2337 for 10 min. Insets are the representative cells. Scale bar = 10 μm. i Flow cytometry showing the percentage of CD71⁺CD235a⁺ cells in HuDEP2 cells after ANXA2 depletion and the statistics (n = 3 biologically independent experiments). j Immunoblotting of p-STAT5 phosphorylation after ANXA2 depletion and the quantitation (n = 3 biologically independent experiments). k Immunoblotting of p-STAT5 phosphorylation following 10 μM CUCPT8m treatment in ANXA2-depleted HuDEP2 cells (n = 3 biologically independent experiments). l Immunoblotting of p-STAT5 phosphorylation following 5 μM CUCPT8m treatment in TLR8 knockout HuDEP2 cells (n = 3 biologically independent experiments). m Normalized cell viability of TLR8 knockout HuDEP2 cells after CUCPT8m treatment for 24 h (n = 3 biologically independent experiments). Data are mean ± SEM; statistical significance was determined using the unpaired two-tailed Student’s t-test for two groups comparison or one-way ANOVA for multiple comparisons. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns = not significant. Source data are provided as a Source Data file.