Fig. 4. NAD⁺ modulation induces premature eryptosis in human erythrocytes.

a Imaging of FITC-dextran-loaded erythrocytes with BSA or rTNT proteins at 2 h post-resealing showed efficient loading of cargo. Protein specific confirmation of loaded erythrocytes with respective antibodies after 48 h of incubation at 37 °C; Scale bar 5 μm. b Evaluation of eryptosis by confocal microscopy images, of protein-loaded erythrocytes stained with FITC-Annexin-V. Scale bar 4 μm. TNT-loaded erythrocytes exhibit enhanced eryptosis. Representative histograms of AnnexinV-binding of erythrocytes after 48 h of incubation are shown. c Histogram of forward scatter representing the decrease in cell volume of rTNT-loaded erythrocytes from control and rIFT-loaded erythrocytes (left). Representative histograms of DCFDA-fluorescence to determine oxidative stress in loaded erythrocytes (middle) are depicted. Representative histograms of Fluo-4AM fluorescence, in rTNT-loaded erythrocytes from control and rIFT-loaded erythrocytes (right) are shown. d Intra-erythrocytic NAD⁺ content in loaded erythrocytes before incubation (at time 0) and after 48 h of incubation at 37 °C. Statistical significance of difference was calculated using unpaired t-tests with Welch’s correction, *P < 0.05, **P < 0.01.