Fig. 2: Erastin combined with KD01 further induces ferroptosis in tumor cells.

A TEM images of mitochondria in SK-OV-3 after 48 h of treatment with Erastin (10 μM), KD01 (MOI 50), or their combination. Red arrows indicate adenovirus particles within the cells. Images were acquired at an accelerating voltage of 80 kV. B MDA levels, a marker of lipid peroxidation, in SK-OV-3 and A2780 cells after 48 h of treatment were detected using the MDA assay kit, based on three biological replicates. Data are presented as means ± SD and were analyzed using Student’s t test. C Intracellular ROS levels in SK-OV-3 and A2780 cells treated as indicated for 48 h were detected using the DCFH-DA fluorescent probe. D The bar plot depicts relative intracellular ROS expression compared to control, based on three biological replicates. Data are presented as means ± SD and were analyzed using Student’s t test. E Mitochondrial ROS levels in SK-OV-3 and A2780 cells after 48 h of treatment were detected using the MitoSOX fluorescent probe. F The bar plot depicts relative mitochondrial ROS levels compared to control, based on three biological replicates. Data are presented as means ± SD and were analyzed using Student’s t test. G Mitochondrial membrane potential (ΔΨm) in SK-OV-3 and A2780 cells treated for 48 h was detected using the JC-1 fluorescent probe. H The bar plot depicts relative changes in mitochondrial membrane potential in SK-OV-3 and A2780 cells, shown as the ratio of JC-1 aggregates to monomers, based on three biological replicates. Data are presented as means ± SD and were analyzed using Student’s t test. I Western blot was performed to detect glutathione peroxidase 4 (GPX4) protein expression in SK-OV-3 and A2780 cells after 48 h of treatment, with GAPDH used as the loading control. Each lane represents samples from a single experiment. J qRT-PCR was used to detect relative mRNA expression of GPX4 in SK-OV-3 and A2780 cells after 48 h of treatment, using GAPDH as the reference gene and analyzed using the 2^−ΔΔCT method relative to control, based on three biological replicates. Data are presented as means ± SD and were analyzed using Student’s t test. K Levels of reduced GSH and GSSG in SK-OV-3 and A2780 cells after 48 h of treatment were detected using the GSH/GSSG assay kit, based on three biological replicates. Data are presented as means ± SD and were analyzed using Student’s t test. L The CCK-8 assay was also used to evaluate the viability of SK-OV-3 and A2780 cells following 48 h of treatment with Erastin (10 μM), KD01 (MOI 50), or their combination, with or without the addition of Fer-1 (50 μM). Data are presented as means ± SD from three biological replicates and were analyzed using Student’s t test. Statistical significance was determined as follows: *p < 0.05, **p < 0.01, ***p < 0.001, NS > 0.05.