Fig. 4

Vimentin degradation moderately enhances the enucleation rate in dTAG-VIM-H9 cells during erythroid differentiation. A Immunofluorescence staining of VIM, GYPA, and DAPI was conducted in cultured H9 or dTAG-VIM-H9 erythroblasts on the 14th day. Scale bars = 10 μm (60X). B Western blot analysis of dTAG-VIM protein (∼ 70.5 kDa) at various time points (2 h, 6 h, 12 h, 24 h and 48 h) following treatment with 0.5 µM dTAG-7 (Cropped blots). Source blots are presented in Additional file 2: Fig. S9. C Flow cytometric analysis of apoptosis of dTAG-VIM-H9 cells induced for 14 days of erythroid differentiation following 48 h of treatment with 0.5 µM dTAG-7, using annexin V and propidium iodide (PI). D Quantitative analysis of (C). Data was obtained from three independent experiments. E Quantitative analysis of CD235⁺CD71⁻ cells on the 14th day of erythroid culture by flow cytometry. Data were obtained from three independent experiments (Related to Fig. S4B). F Flow cytometric analysis of erythroid markers and nuclear dye (CD235a, Hoechst33342) on 16th day of dTAG-VIM-H9 erythroid culture. The CD235a⁺Hoechst⁻ population represents enucleated erythrocytes. G Statistical analysis of enucleated erythrocytes following treatment with 0.5 µM dTAG-7. Data was obtained from three independent experiments (Related to F)