Hello Chien-Chih Chiu

I suggest it could be autophagy-associated cell death. RPE cells are known to undergo such type of cell death due to oxidative stress. You can check with the marker for autophagy namely, Beclin-1.

I can see that the cell is swollen, but is it going to die or repairing itself?

Best Wishes.

You can not be sure just by looking at the morphology, but you could check for lipid peroxidation, e.g. with BODIPY dyes and flow cytometry, and related ferroptosis. Also, autophagy might play role, which can be detected via LC3-I/II and appropriate inhibitors for autophagic flux analysis.

Hope that helps a bit - CL

This is may be apoptosis or necrosis

Article Hydrogen Peroxide-Induced Cell Death in a Human Retinal Pigm...

Hydrogen peroxide significantly induced cell death in ARPE-19 cells, whereas pretreatment of the cells with catalase prevented cell death. Application of the ISEL assay and acridine orange/ethidium bromide staining demonstrated that the H2O2-induced cell death occurred by an apoptotic mechanism at lower concentrations of H2O2 (400, 500, 600 microM), whereas higher concentrations of H2O2 induced necrosis rather than apoptosis. Caspase 3 was associated with the apoptotic pathway in human RPE cell death. Western blot analysis confirmed caspase 3 activation and cleavage of substrate proteins in ARPE-19 cells treated with an H2O2 concentration of 600 microM. These results indicate that treatment with H2O2 induces apoptotic and necrotic cell death in ARPE-19, and that caspase 3 is associated with apoptotic cell death. Therefore, H2O2 may induce the destruction of RPE cells in AMD by the combined effects of apoptosis and necrosis.

https://www.nature.com/articles/cddis2013478

The purpose of this study is to systematically dissect the mechanism of RPE cell death induced by oxidative stress. Our results show that characteristic features of apoptosis, including DNA fragmentation, caspase 3 activation, chromatin condensation and apoptotic body formation, were not observed during RPE cell death induced by either hydrogen peroxide or tert-Butyl hydroperoxide. Instead, this kind of cell death can be prevented by RIP kinase inhibitors necrostatins but not caspase inhibitor z-VAD, suggesting necrotic feature of RPE cell death. Moreover, ATP depletion, receptor interacting protein kinase 3 (RIPK3) aggregation, nuclear and plasma membrane leakage and breakdown, which are the cardinal features of necrosis, were observed in RPE cells upon oxidative stress. Silencing of RIPK3, a key protein in necrosis, largely prevented oxidative stress-induced RPE death. The necrotic nature of RPE death is consistent with the release of nuclear protein high mobility group protein B1 into the cytoplasm and cell medium, which induces the expression of inflammatory gene TNFα in healthy RPE and THP-1 cells. Interestingly, features of pyroptosis or autophagy were not observed in oxidative stress-treated RPE cells. Our results unequivocally show that necrosis, but not apoptosis, is a major type of cell death in RPE cells in response to oxidative stress. This suggests that preventing oxidative stress-induced necrotic RPE death may be a viable approach for late-stage dry AMD.

Main

The morphology looks like cells are going through senescent and/or autophagy. Perhaps how long of the treatment and whether the cells were starved during the treatment does play an important factor. Checking on autophagy has many methods, and one of the easy method is to stain the cells with acridine Orange to see the number of acidic vesicles organelles as autophagy involved lysosome.

i recommend to you this paper : Frédéric Ziegler, Lamia Seddiki, Rachel Marion-Letellier, Alain Lavoinne, Pierre Déchelotte,

Effects of l-glutamine supplementation alone or with antioxidants on hydrogen peroxide-induced injury in human intestinal epithelial cells,

e-SPEN, the European e-Journal of Clinical Nutrition and Metabolism.

doi: 10.3892/ijmm.2012.1215. Epub 2012 Dec 18.. 2013 Feb;31(2):471-6.Int J Mol Med. The effects of exogenous H2O2 on cell death, reactive oxygen species and glutathione levels in calf pulmonary artery and human umbilical vein endothelial cells.

Can be useful

An important point is probably the time course of your death process, concerning necrosis it usually takes 2-3 hours to be completed and could be followed by trypan blue accumulation within the cells; in my experience, apoptosis is a slower process (24-30 h) and can be blocked by inhibitors of protein synthesis such as cycloheximide. You can also use Dapi staining to observe the formation of nuclear bodies.