Fig. 5

Application of the single-vector dCasRx translation enhancement tool in a CaOx crystal-induced cell injury model. The HK2 cell lines were stably transfected with lentiviral vectors for FTH1 overexpression (LV-FTH1), the dual vector CRISPR-dCasRx-eIF4GI tool targeting FTH1 (DV-FTH1), the single-vector CRISPR-dCasRx-eIF4GI tool targeting FTH1 (SV-FTH1). A The mRNA expression level of FTH1 was detected using qRT–PCR analysis for four groups. B The protein expression of FTH1 was verified using western blot and the bar graph shows the relative expression level of FTH1. C The cell viability was detected by CCK8 assay kit. D Two indicators of ferroptosis, including MDA content and Fe²⁺ content, were measured. E The degree of lipid peroxidation was measured using the flow cytometric analysis in four groups and the bar graph shows the mean fluorescence intensity. F Immunofluorescence analysis was performed to determine the expression of FTH1 (magnification ×400) and the bar graph shows relative protein levels. G Deferoxamine (DFO) is an iron chelator and a ferroptosis inhibitor. The 50 μM and 100 μM concentration of DFO is used. Two indicators of ferroptosis, including Fe²⁺ content and MDA content, were measured. H The level of cellular Fe²⁺ was observed and images were taken under dark field (magnification ×400). Brighter red indicates higher levels of cellular Fe²⁺ and the bar graph shows the mean fluorescence intensity. I The degree of lipid peroxidation was observed and images were taken under dark field (magnification ×400). Higher ratio of green to red indicates higher degree of lipid peroxidation. Data are presented as the means ± SEM from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, and ns, no significant difference, represents P > 0.05