Ferroportin-Dependent Iron Homeostasis Protects against Oxidative Stress-Induced Nucleus Pulposus Cell Ferroptosis and Ameliorates Intervertebral Disc Degeneration In Vivo

Figure 4

TBHP treatment aggravated FPN downregulation in human NPCs by MTF1 suppression. (a, b) The human NPCs were treated with DMSO or TBHP (50 μM) for 12 h. The quantitative values are expressed as (); . (a) MTF1 protein levels in cytosolic and nuclear fractions of human NPCs were determined using western blotting, which was, respectively, normalized to GADPH and Histone 3. (b) The human NPCs were fixed and stained with DAPI (nuclear) and MTF1 antibody. Representative images were shown to visualize the subcellular locations of MTF1. (c) The subcellular localization of MTF1 was performed in human NP tissues from the control (NC) groups and IVDD groups by immunohistochemistry analysis. (d, e) Lenti-Vector or Lenti-MTF1 infection was performed in human NPCs. The quantitative values are expressed as (); . (d) MTF1 protein levels in cytosolic and nuclear fractions of human NPCs were determined using western blotting, which was, respectively, normalized to GADPH and Histone 3. (e) Representative western blotting assay and quantitation of the level of MTF1, FPN, and FTL proteins, which was normalized to β-actin. (f–i) Lenti-Vector or Lenti-MTF1 infection and scrambled siRNA (siCON) or FPN siRNA (siFPN) transfection were performed before DMSO or TBHP treatment (50 μM) for 12 h. Data are expressed as (); . (f) Intracellular total free iron levels in human NPCs were assayed using iron assay kit. (g, h) Lipid ROS levels were assayed using C11-BODIPY 581/591 using flow cytometry. (i) Cell viability was examined by the absorbance of CCK-8.

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