Figure 3. Activation of the UPS is critical for mitophagy during human erythropoiesis.

(A) Representative Western blot analysis and quantification of ribosomal proteins (RPL29), rapidly removed mitochondrial proteins (RRMPs) and slowly removed mitochondrial proteins (SRMPs) in proerythroblasts collected at different stages of terminal erythroid maturation. (n=3). (B) Representative Western blot analysis of RPL29, RRMPs and SRMPs in proerythroblasts differentiated in the presence of UPS (MG132 or bortezomib) or autophagy (Bafilomycin A1 or E-64d/Pepstatin) inhibitors. (C) Representative immunofluorescence images showing colocalization of the lysosomal maker LAMP1 with COXIV (RRMP) or ATP5A (SRMP) in proerythroblasts collected at different stages of terminal erythroid maturation. White arrowheads indicate colocalization. Scale bar = 2 μm. (D) Relative chymotrypsin-like UPS activity over time during proerythroblast maturation. (n=5). (E) Autophagy flux over time during proerythroblast maturation. (n=3). (F) Western blot analysis and quantification of COXIV and ATP5A levels in HD and CANDLE proerythroblasts 24 h and 144 h post-differentiation. (HD – Healthy donor). (G) Representative FACS plot of Mitotracker Deep Red (MTDR) staining on mature RBCs (gated on CD235a⁺ CD71⁻ cells) isolated from CANDLE3 patient. (H) Western blot analysis of selected mitochondrial proteins in proerythroblast transfected with short hairpin RNAs (shRNAs) specific for 15- Lipoxygenase (ALOX15). In (B, C and H) results are representative of at least three independent experiments. Data are means ± SEM. [One-way analysis of variance (ANOVA) with Tukey post hoc test for multiple comparisons in (A), (D) and (E)].