Fig. 1: ARPE-19 cells treated with SI manifest necrotic phenotype.

A Cell viabilities determined by CCK assays after NaIO₃ treatment for 12 h or 24 h at indicated concentrations (n = 4). B ARPE-19 cells were cultured with NaIO₃ at indicated concentrations for 24 h. After the treatment, the LDH levels were determined by the LDH assays (n = 4). C Left: Fluorescence images of cells stained with JC-1, an indicator of mitochondrial membrane potential (MMP). ARPE-19 cells were treated with 10 mM NaIO₃ for 24 h. Scale bar: 100 μm. Right: The ratio of red/green fluorescence intensity shows the change of MMP (n = 3). D ARPE-19 cells treated with 10 mM NaIO₃ for 24 h were observed by scanning electron microscope. Scale bar: 20 μm. E Upper: Fluorescence images of cells treated with 10 mM NaIO₃ for 24 h followed by Hoechst 33342 and PI double staining. Scale bar: 100 μm. Lower: Cells positively stained with Hoechst and PI were counted using Image J software. Graphs represent the percentage of Hoechst and PI stained cells against total cells (n = 3). F Fluorescence-activated cell sorting (FACS) histograms of Annexin V-FITC/PI stained cells with (right) and without (left) the treatment of 10 mM NaIO₃ for 24 h (Q1: cellular debris or necrotic cells; Q2: necrotic or late apoptotic cells; Q3: early apoptotic cells; Q4: viable cells). G Left: Effect of the caspase inhibitor Z-VAD on ARPE-19 cell death. Cells were pretreated with Z-VAD or solvent alone (DMSO) at indicated concentrations for 3 h and followed by co-treatment with or without 20 mM NaIO₃ for 24 h (n = 4). Right: A positive control for apoptosis inhibition by Z-VAD. Jurkat cells were pretreated with 100 μM Z-VAD or solvent alone (DMSO) for 3 h followed by co-treatment with 1.5 μM doxorubicin (DOX) for 18 h (n = 4). H Left: TUNEL staining of apoptotic ARPE-19 cells. Cells were exposed to 10 mM NaIO₃ for the indicated time. Scale bar: 100 μm. Right: numbers of apoptotic cells and normal cells in the microscope field of view were counted by image J software (n = 3).