Figure 1.

(A) Schematic representation of the experimental setup. Wildtype (WT) and Pink1^−/− mouse embryonic fibroblast (MEF) were either left untreated or incubated with ferric ammonium acid (FAC), DFO (Deferoxamine), and 2,2′-Bipyridine (22BP). mRNA expression was analyzed by RT-qPCR after 16 h incubation, while protein abundances were analyzed by mass spectrometry and quantitative immunoblots after 48 h incubation. The FAC treatment is highlighted in orange, whereas the two iron chelator treatments are highlighted in light and dark blue, as the color code for the entire manuscript. (B) Summary of mass spectrometry results for the seven conditions studied in comparison, showing the total number of detected proteins and the number of factors with significantly changed abundance, with the respective cutoff values for the significance and the fold-change (shown as log₂ difference). The analysis of WT 22BP versus WT ctrl. and of Pink1^−/− 22BP versus Pink1^−/− ctrl. revealed so many significant factors that a log₂ difference of 1.0 was used as the cutoff for downstream pathway enrichment analyses. (C) As measures of quality control for the culture incubations, the fold-changes as log₂ differences are shown for well-established iron homeostasis factors, as detected in mass spectrometry, in comparison to (D) the respective log₂ differences by RT-qPCR for such key iron homeostasis genes. The RT-qPCR results were normalized to Tbp expression levels (Tata-binding protein encoding mRNA). ctrl. = untreated control condition.