Quantitative analysis of autophagic flux reveals radiation-induced activation of SQSTM1-mediated degradation of protein aggregates and ER-phagy.

Autophagy is an evolutionarily conserved process that degrades and recycles intracellular components through lysosomes, thereby maintaining cellular homeostasis under stress conditions. Although radiation is known to influence autophagy, most previous studies have relied on static marker expression rather than quantitative evaluation of autophagic flux. In the present study, we quantitatively analyzed autophagic flux in hTERT/RPE-1 cells exposed to γ-rays (0.5-4 Gy) using both bafilomycin A1-based assays and HaloTag reporter systems that visualize lysosomal degradation. LC3-based total autophagic flux remained unchanged even at 4 Gy, indicating that lysosomal function is preserved after irradiation. In contrast, SQSTM1-dependent selective autophagy increased significantly at doses of 2 Gy or higher, suggesting enhanced clearance of radiation-induced protein aggregates. HaloTag-based analyses further revealed that γ-irradiation induced mitophagy and ER-phagy in a dose-dependent manner, consistent with activation of oxidative stress and unfolded protein response pathways. These findings demonstrate that ionizing radiation does not globally suppress autophagy but selectively activates organelle-specific autophagy, particularly SQSTM1-mediated ER-phagy. The selective activation of these quality-control pathways likely contributes to maintaining cellular integrity and stress adaptation following irradiation. Quantitative flux analysis thus provides new insight into the hierarchical regulation of autophagy and its role in cellular survival and repair mechanisms after radiation exposure.