Fig. 5

Targeted vimentin degradation promotes nuclear polarization in preparation for enucleation during erythroid differentiation. A Representative images of dTAG-VIM-H9 cells cultured for 16 days in erythroid differentiation, stained with CD71-APC, CD235a-FITC (GYPA) and Hoechst33342 (HO). Left: no treated, Right: 0.5 µM dTAG-7 treated. Scale bar = 7 μm. B Representative image of nuclear polarization, where the Delta Centroid XY parameter represents the distance between the cell body center and the nucleus center. C The degradation of VIM protein mediated by dTAG-7 resulted in the enhancement of nuclear polarization, as evidenced by the distribution of the parameter Delta Centroid BF-HO. This parameter measures the distance between the center of the cell body observed in bright-field microscopy and the center of the nuclear staining achieved with Hoechst33342 (HO). The median and mean values of the Delta Centroid BF-HO of control and dTAG7-treated dTAG-VIM-H9 erythroblasts are presented, and the difference between the two samples is statistically significant (P < 0.0001). D Representative images of typical cells classified as polarized (cell containing a condensed nucleus located to one side, close to the plasma membrane. Red circle), centered (nucleus localized at the center of the cells. Green circle), and others (multinucleate or non-erythroid cells. Sky blue circle). Cells are stained with Benzidine and Giemsa reagent. Scale bar = 10 μm. E Statistical analysis of the proportion of dTAG-VIM-H9-derived erythroid cells undergoing nuclear polarization under culture conditions with or without 0.5 µM dTAG-7. Erythroid cells derived from H9 served as a WT control. Data were obtained from three independent replicate experiments (Related to D)