Fig. 3: The effect of drugs on ferroptosis in CVB3-infected cells.

A Cell viability of HeLa cells in different drug treatment groups measured by CCK-8 assays. Fer-1 pretreated at 2 μM, 24 h before CVB3 infection. DFO pretreated at 5 μM 24 h before CVB3 infection. DMSO was used as a solvent control. The results showed that both Fer-1 and DFO could prevent the decrease in cell viability caused by CVB3 infection. B Area of CVB3-positive fluorescence measured with ImageJ, which corresponds to figure E. C MDA detection under different ferroptosis drug interventions. It showed that both Fer-1 and DFO drugs can reduce MDA production, especially DFO (p < 0.01). D Iron concentrations were measured after pretreatment of CVB3-infected HeLa cells with the ferroptosis agonist Erastin (6 μM, 12 h) and inhibitors Fer-1 and DFO. E CVB3 fluorescence (green) determined by IFA in HeLa cells in different drug intervention groups. Cell nuclei were stained with DAPI (blue) (scale bar = 50 μm). F Fluorescence of lipid ROS in HeLa cells pretreated with Fer-1 or DFO and incubated with the BODIPY581/591C11 probe (scale bar = 100 μm). G Percentage of oxidized/non-oxidized cells were calculated by ImageJ software according to figure F. It suggested that both DFO and Fer-1 could decrease the oxidized lipid ROS significantly (p < 0.001). H Flow cytometry was used to detect lipid ROS in different drug pretreatment groups during CVB3 infection. I, J The protein level of ACSL4 in drug groups were measured by western-blot, the gray values of strips were calculated by ImageJ. K, L The protein level of GPX4 in different drug groups were measured by western-blot. Measurement of relative gray values indicating gene protein levels were determined using ImageJ. M, N The mRNA level of ACSL4 and GPX4 in the drug-pretreated groups were detected via qPCR. All results are expressed as the mean ± SD. *p < 0.05 vs. the mock group, **p < 0.01, ***p < 0.001. #p < 0.05 vs. the CVB3 group, ##p < 0.01, ###p < 0.001. n = 3.