Single-cell analysis reveals loss of eTNS1 impairs erythroblast enucleation. (A) Schematic of erythroblast classification from eTNS1-KO erythroid cultures into 4 categories based on Hoechst (nuclei; blue), GPA (green), and eTNS1 (orange) staining. Cells were classified as eTNS1⁺ erythroblasts, eTNS1⁻ erythroblasts, eTNS1⁺ reticulocytes, and eTNS1⁻ reticulocytes. Figure created with Biorender.com. (Diaz, D. 2025, https://app.biorender.com/illustrations/655d7217e036ccb922863c17). (B) Representative single tiled image showing the heterogeneous cell population from an eTNS1 CRISPR KO culture using the Zeiss CD7. Scale bar, 100 μm. (C) Representative higher magnification images of individual cells in panel A depicting the 4 classes: (a) eTNS1⁺ erythroblast, (b) eTNS1⁻ erythroblast, (c) eTNS1⁺ reticulocyte, and (d) eTNS1⁻ reticulocyte. Scale bar, 5 μm. (D) Quantification of GPA intensity levels of eTNS1⁺ and eTNS1⁻ cells from day-14 and day-17 eTNS1-KO cultures; 3 to 5 tiled images were taken from a single coverslip for each culture. Plot reflects the mean ± SD of 1500 eTNS1⁺ and eTNS1⁻ erythroblasts from 3 independent eTNS1-KO cultures. (E) Quantification of eTNS1⁻ and eTNS1⁺ reticulocytes in control, nontarget, and KO day-14 and day-17 cultures. Percentage reticulocytes calculated by dividing the number of reticulocytes by (the number of reticulocytes plus the number of erythroblasts). Plot reflects mean ± SD of percentage reticulocytes calculated from ∼6000 cells for each experiment. Three independent experiments performed for each condition. ∗∗∗P = .0002; ∗∗∗∗P < .0001. ns, non-significant. Schematic in panel A created with BioRender.com.