Fig 1: Excess iron induces differential expression of stress and autophagy proteins. (A) Human hepatoma HepG2 cells, mouse AML12 hepatocytes, and mouse macrophage-like RAW264.7 cells, and (B) primary human aortic endothelial cells (HAECs) and primary mouse hepatocytes (P-Hepa) were treated with 100 µM FAC for the indicated times. The expression of stress proteins (NRF2, ATF4, CHOP, Grp78, and phospho-PERK), autophagy markers (p62 and LC3B), and ferritin heavy chain 1 (FTH1, a maker of iron loading) was determined by Western blotting. Numbers below the blots represent mean ± SE (n = 3 independent experiments), with the untreated control set at 100. For LC3B protein, the active isoform LC3B-II was quantified. (C) C57BL/6 mice were injected (i.p.) with iron dextran (FeDex, 500 mg/kg/b.w.) or PBS as a control. After 24 h, livers were harvested for the analysis of the expression of stress and autophagy proteins. Data are mean ± SE (n = 6 mice of two independent experiments). *, P < 0.01; and **, P < 0.05 compared to the control.
Fig 2: ATF4 deficiency leads to iron-dependent autophagic dysfunction, mtROS production and ferroptosis. (A) 293 T cells with stable knockdown of ATF4 (shATF4) or transduction with scrambled shRNA (shCont). (B) 293 T/shATF4 and shCont cells transiently expressing GFP-LC3-RFP were treated with or not 100 µM FAC for 24 h. Representative images are shown (left). The numbers of yellow puncta were quantified, and the percentages of yellow (white arrow) vs the total numbers of puncta are presented as mean ± SE (n = 15 fields of three independent experiments). (C) LC3B-II expression in 293 T/shATF4 and shCont cells that were treated with or not FAC for 24 h. Data shown are mean ± SE (n = 3 independent experiments). (D-E) 293 T/shATF4 and shCont cells were treated with or not 100 µM FAC or 5 µM Fer-1. After 24 h, cells were then stained with MitoSOX or PI. Relative fluorescence intensity (RFI) of MitoSOX staining per field was analyzed by ImageJ, and the percentage of PI + cells over the total numbers of cells per field was quantified. Data are mean ± SE (n = 9 fields of three independent experiments). *, P < 0.01 compared to untreated shCont cells; &, P < 0.01 relative to untreated shATF4 cells; and #, P < 0.01. Bars, 50 µm. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
Fig 3: Iron deficiency induces ER stress response and attenuates autophagic flux in in vitro. (A) AML12, RAW264.7 and P-Hepa were treated with 100 µM DFO for the indicated times. The expression of stress proteins (NRF2, ATF4, CHOP, and phospho-PERK), autophagy markers (p62 and LC3B), and FTH1 was determined by Western blotting. Data are mean ± SE (n = 3 independent experiments), with the untreated control set at 100. For LC3B protein, the active isoform LC3B-II was quantified. Arrowhead denotes the upper bands are phospho-PERK (p-PERK). (B) Protein expression of ER stress and autophagy factors in DFO-treated HepG2 cells. (C) AML12 cells were treated with 100 µM DFO for 48 h in the presence or not of 100 µM chloroquine (Chlq) during the last 4 h. Changes in LC3B-II expression were quantified relative to untreated control (set at 100). Data are mean ± SE (n = 3 independent experiments). (D) The expression of cleaved caspase-3 (CC3) in DFO-treated AML12 cells. Data are mean ± SE (n = 3). *, P < 0.01; and **, P < 0.05 compared to the untreated control. n. s., not significant.
Supplier Page from DNASU for ATF4 (Homo sapiens) in pLX304 (Gateway V5-tagged lentiviral expression vector)