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. 2019 Nov 14;4(22):e130111. doi: 10.1172/jci.insight.130111

Figure 4. Bitopertin treatment modulates HRI activity and improves autophagy, resulting in optimization of the quality control process with amelioration of red cell survival.

Figure 4

(A) Wb analysis of HRI phosphorylation and eIF2αP levels in sorted 1.5 × 106 CD44+Ter119+FSChi spleen cells from Hbbth3/+ mice with and without bitopertin treatment (10 mg/kg/d, 28 days) (n = 3). (B) Left: Wb analysis of phospho-mTOR (p-mTOR), mTOR, Rab5, Lamp1, and p62 in reticulocyte-enriched fractions from WT and Hbbth3/+ mice with and without bitopertin treatment (n = 3; 10 mg/kg/d, 28 days). Catalase was used as a protein loading control. Right: Densitometric analyses of the immunoblot bands similar to those shown are presented. Data are shown as median and minimum/maximum (n = 4); *P < 0.05 compared with WT; °P < 0.05 compared with vehicle; 2-way ANOVA with Holm-Šídák test for multiple comparisons. (C) Hemichromes bound to the membrane in WT and Hbbth3/+ mice with and without bitopertin treatment (n = 3; 10 mg/kg/d, 28 days). Data are shown as median and minimum/maximum (n = 4); P < 0.05 compared with WT; °P < 0.05 compared with vehicle; 2-way ANOVA with Holm-Šídák test for multiple comparisons. (D) ROS levels in red cells of WT and Hbbth3/+ mice with and without bitopertin treatment (10 mg/kg/d, 28 days). Data are presented as median and minimum/maximum (n = 6); *P < 0.05 compared with vehicle-treated Hbbth3/+mice; °P <0.05 compared with vehicle. 2-way ANOVA with Holm-Šídák test for multiple comparisons. (E) Wb analysis of thioredoxin-reductase (Trx-R) and peroxiredoxin-2 (Prx2) in erythrocytes from WT and Hbbth3/+ mice with and without bitopertin treatment (n = 3; 10 mg/kg/d, 28 days). Catalase was used as a protein loading control (see Supplemental Figure 6, A and B, for densitometric analyses). (F) Red cell survival of CFSE-labeled red cells from WT and Hbbth3/+ mice with and without bitopertin treatment (n = 3; 10 mg/kg/d, 40 days). Data are presented as mean ± SD (n = 3) from each group; *P < 0.05 compared with WT; °P < 0.05 compared with vehicle; Wilcoxon’s signed-rank test for multiple comparisons.