Abstract: Objective To develop a modified lentiviral expression system of mCherry-GFP-LC3B, driven by the hematopoietic-specific SFFV (spleen focus-forming virus) promoter, in order to perform an efficient and real-time monitoring of autophagy flux with in terminally differentiated erythroid cells. Methods The lentiviral plasmid pRSC-SFFV-mCherry-GFP-LC3B was constructed and packaged into lentiviruses for infection of human erythroid progenitor cell line(HUDEP-2) and mouse fetal liver-derived primary erythroid cells. Autophagy dynamics of the cells were then analyzed using fluorescence imaging, Western blot, and flow cytometry in serum starvation and chloroquine inter-vention models. Results The SFFV promoter rendered significantly higher reporter expression efficiency than CMV promoter in HUDEP-2 (97% vs. 60%)(P＜0.01) and in the primary mouse erythroid cells (83% vs. 1%)(P＜0.001), without disrupting normal erythroid differentiation. Serum deprivation increased autolysosomes (red puncta), elevated LC3-Ⅱ/LC3-Ⅰ ratios, and decreased p62 levels. Chloroquine treatment induced autophagosome (yellow puncta) accumulation (P＜0.001), showing a dose-dependent inhibition of autophagy flux (r²=0.92). Conclusions The SFFV-driven dual-fluorescent system enables robust and real-time monitoring of autophagy flux in erythroid cells, providing a sensitive tool for mechanistic study of erythroid differentiation and related disorders such as anemia.

Key words: SFFV promoter, autophagy, dual-fluorescent mCherry-GFP-LC3B, erythroid cells