Figure 6.
The presence of ER proteins and remnants in RBCs of nonspl patients contrasts with lysosome and mitochondria proteins and remnants in RBCs of spl patients. RBCs from patients, either spl (filled circles or semifilled circles for intrafamily comparison) or nonspl (open circles), and RBCs from CTLs (dark blue dotted line or CTL ratio or ΔP–CTL) or a healthy splenectomized donor (red dotted line) were compared for membrane association of proteins of the ER-ribosomes (A), lysosomes (B), or mitochondria (C) and for the presence of organelle remnants (D-F). Scale bar represents 5 μm in panel D. Statistics are indicated above the patient cohorts for the comparison with CTL values and above a horizontal line for comparison between the 2 patient cohorts, respectively. (A-C) Membrane ghost association of ribosomal protein S25 (RPS25), lysosomal-associated membrane protein 1 (LAMP1) and ATP synthase F1 subunit alpha (ATP5F1A), respectively, enriched in ribosomes, lysosomes, and mitochondria and determined by proteomics (statistical analysis and additional examples in supplemental Figure 3). (D-F) Organelle labeling. RBCs spread on PLL-coated coverslips were labeled with BODIPY-ceramide (Cer), LysoTracker, or MitoTracker and observed by fluorescence microscopy (LysoTracker was maintained during observation). (D) Representative images. Open and filled arrowheads, patches, and network-like structures, respectively. (E-F) Quantification of RBCs presenting LysoTracker- or MitoTracker-positive patches expressed as percentage of the total RBC population and then as ΔP–CTL (mean of 1-3 and 1-7 independent experiments perpatient in panels E and F, respectively; Kruskal-Wallis tests followed by Dunn post hoc). (G-L) Membrane ghost association of band 4.2 (EPB42), Rh-associated glycoprotein (RhAG), voltage-dependent anion channel 1 (VDAC1), translocator protein (TSPO), Transferrin receptor (TfR) and DNM2 (dynamin), respectively, involved in cytoskeleton anchorage complexes (G-H), mitophagy (I-J), and endocytosis (K-L) and determined by proteomics (for volcano plots, see supplemental Figure 3).

The presence of ER proteins and remnants in RBCs of nonspl patients contrasts with lysosome and mitochondria proteins and remnants in RBCs of spl patients. RBCs from patients, either spl (filled circles or semifilled circles for intrafamily comparison) or nonspl (open circles), and RBCs from CTLs (dark blue dotted line or CTL ratio or ΔP–CTL) or a healthy splenectomized donor (red dotted line) were compared for membrane association of proteins of the ER-ribosomes (A), lysosomes (B), or mitochondria (C) and for the presence of organelle remnants (D-F). Scale bar represents 5 μm in panel D. Statistics are indicated above the patient cohorts for the comparison with CTL values and above a horizontal line for comparison between the 2 patient cohorts, respectively. (A-C) Membrane ghost association of ribosomal protein S25 (RPS25), lysosomal-associated membrane protein 1 (LAMP1) and ATP synthase F1 subunit alpha (ATP5F1A), respectively, enriched in ribosomes, lysosomes, and mitochondria and determined by proteomics (statistical analysis and additional examples in supplemental Figure 3). (D-F) Organelle labeling. RBCs spread on PLL-coated coverslips were labeled with BODIPY-ceramide (Cer), LysoTracker, or MitoTracker and observed by fluorescence microscopy (LysoTracker was maintained during observation). (D) Representative images. Open and filled arrowheads, patches, and network-like structures, respectively. (E-F) Quantification of RBCs presenting LysoTracker- or MitoTracker-positive patches expressed as percentage of the total RBC population and then as ΔP–CTL (mean of 1-3 and 1-7 independent experiments perpatient in panels E and F, respectively; Kruskal-Wallis tests followed by Dunn post hoc). (G-L) Membrane ghost association of band 4.2 (EPB42), Rh-associated glycoprotein (RhAG), voltage-dependent anion channel 1 (VDAC1), translocator protein (TSPO), Transferrin receptor (TfR) and DNM2 (dynamin), respectively, involved in cytoskeleton anchorage complexes (G-H), mitophagy (I-J), and endocytosis (K-L) and determined by proteomics (for volcano plots, see supplemental Figure 3).

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