Fig. 4.

GPx3 knockdown increases cell death and levels of extracellular H₂O₂ in response to high dose ascorbate. A: IC50 values of ascorbate on ovarian cancer cells were determined by cell viability assay. OVCAR3 shGPx3 stable cell lines (5 × 10³/well) were seeded into 96-well plates and exposed to various concentrations of ascorbate for 24 h. Cell viability was determined by crystal violet uptake and IC50 values calculated as shown. B: GPx3 scavenges ascorbate-induced H₂O₂. Cells (5 ×10³/well) were seeded into 96-well plates and treated with ascorbate for 24 h. Medium from each well was collected and immediately analyzed using Amplex Red·H₂O₂ concentrations were derived from standard curves and data expressed as mean ± SEM (n = 3/group; Two-way ANOVA, Tukey's post hoc, ****p < 0.0001). C: The effects of GPx3 knockdown on cell viability were assessed under conditions of anchorage independence by culturing cells in ULA plates and treated with 0.3 mM ascorbate for 48 h, followed by staining with calcein-AM to indicate live cells (green), and Ethidium homodimer-1 to indicate dead cells (red; Bottom image overlay). Representative images are shown. Scale bar = 100 µm. D: Quantification of live/dead cell fractions in anchorage-independent OVCAR3 spheroids following 48 h treatment with indicated doses of ascorbate. Data expressed as mean ± SEM form 3 independent experiments (n = 4–12/group; one-way ANOVA, Tukey's post hoc, * p < 0.05, ****p < 0.0001).