L-cit suppresses ferritinophagy to ameliorate FAC-induced cytoplasmic ROS accumulation in the mTEC1 cells. (A) Determination of siNCOA4 concentration. mTEC1 cells were transfected with negative control siRNA or different concentration of siNCOA4 to detect the NCOA4 mRNA level. (B) Confirmation of siNCOA4 concentration by qPCR and NCOA4 protein quantitation by western blotting after transfection with 50 nM siNCOA4. The mTEC1 cells were transfected with 50 nM siNCOA4, followed by 2 mM L-cit, and treated with 200 μM FAC for 24 h. (C,D) Immunoblot analyses of Nrf2 in cytoplasm. GAPDH were used as the loading control. (C,E) Immunoblot analyses of Nrf2 in nucleus. H3 was used as the loading control. (F) ROS mean fluorescence intensity. (G) Cytoplasmic ROS level. Values represent mean ± SEM. of three independent experiments. Significant differences are represented by * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns (not significant).