Figure 4.
Ferritinophagy induced by NDV contributes to ferroptosis initiation
(A) Western blot analysis of the levels of LC3 and NCOA4 in NDV-infected U251 cells. Baf-A1 was used as an autophagy inhibitor. β-actin was used as the loading control.
(B) Fluorescence microscopy was used to colocalize NCOA4 (red) and LC3 (green) in U251 cells. Rapamycin was used as an autophagy inducer. Scale bars = 20 μm.
(C) Western blotting analysis of the expression levels of TFR1 in NDV-infected U251cells. Western blotting samples corresponding to the marked timepoints were collected and analyzed.
(D and E) FTH1 and NCOA4 expression levels in NCOA4-knockdown cells. Cells were treated for 24 h with or without NDV. Data are representative of three experiments.
(F) Expression levels of Reactive oxygen species were observed in U251 cells with NCOA4 knockdown with fluorescence microscopy, Erastin was used as a ferroptosis inducer.
(J and H) Expression levels of ferrous iron in NCOA4-knockdown cells were observed with confocal fluorescence microscopy, Erastin was used as a ferroptosis inducer.
(I) Cell death was examined with an LDH assay 24 h after pretreatment with or without DFO.
(J) A model of the possible mechanisms underlying the induction of ferroptosis by NDV through nutrient deprivation and ferritinophagy in U251 cells.
Significance was analyzed using a two-tailed Student's t-test. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. Data were expressed as mean SEM (n = 3 in each group).