ROS is a key mechanism that induces paraptosis. ROS generation was increased in the lens epithelium of patients with early ARCC (
Supplementary Fig. S1). To explore whether ROS serves as a potential mechanism for paraptosis in LECs of early ARCC, we treated LECs with different concentrations of H
2O
2. After a 24-hour exposure of LECs to H
2O
2 from 25 to 400 µM, compared to the control group, ALIX expression decreased with the 50-µM H
2O
2 treatment, while GRP78 expression increased. The BAX/BCL-2 expression ratio showed no significant difference (
Figs. 2A,
2B). However, at a concentration of 200 µM H
2O
2, the BAX/BCL-2 ratio significantly increased (
Figs. 2A,
2B). TEM observations revealed extensive vacuolization of the ER in cells exposed to 50 µM H
2O
2 compared to control cells (
Fig. 2C). To further investigate the origin of vesicles induced by 50 µM H
2O
2, we conducted live-cell staining with fluorescent trackers targeting the ER, mitochondria, lysosomes, autophagosomes, endosomes, and macropinosomes. Interestingly, the vesicles exclusively colocalized with ER fluorescence, without colocalization with any other trackers (
Figs. 2D,
2E). Additionally, treatment with 50 µM H
2O
2 significantly increased the paraptosis-related protein levels of GRP78, p-ERK, and p-P38, whereas ALIX expression inversely decreased (
Figs. 2F,
2G). The morphology and protein expression in cells treated with 50 µM H
2O
2 were consistent with those observed in cells treated with the paraptosis inducer, WA (
Supplementary Fig. S2). To determine whether apoptosis was involved in the cell death of LECs mediated by 50 µM H
2O
2, we pretreated the LECs with ZVAD-FMK, a pan-caspase inhibitor. ZVAD-FMK failed to reduce cell death induced by 50 µM H
2O
2; however, it significantly decreased cell death caused by 200 µM H
2O
2, although not eliminating it (
Fig. 2H). Taken together, these data indicate that ROS induces paraptosis in LECs.