Fig 1: LRRK2G2019S RAW264.7 macrophages show changes in iron homeostasis. (A) Western blot analysis on whole-cell lysates of pRab8T72 and pLRRK2S1292 in wild-type (WT) macrophages, LRRK2G2019S macrophages (control, ‘C’) and LRRK2G2019S macrophages treated with 1 μM MLi-2 for 16 h. Vinculin serves as the loading control. (B) Densitometry analysis of pRab8:Rab8 and pLRRK2:LRRK2 ratios from WT or LRRK2G2019S (‘GS’) macrophages, with and without 1 μM MLi-2 treatment. The mean±s.d. of normalized band intensities from three biological replicates are shown. (C) Volcano plot showing differentially expressed proteins in LRRK2G2019S compared to WT macrophages. On the x-axis, fold change (FC), and, on the y-axis, −log10[adjusted statistical significance (Padj)] are plotted. Dashed lines represent the cut-offs for analysis; proteins with |log2FC|>1 and Padj>1.3 (P<0.05) were considered as differentially expressed. Red dots depict some of the differentially expressed proteins important for iron metabolism. (D) Heat map and table depicting the fold change and the role of the main iron-related proteins differentially expressed in LRRK2G2019S macrophages. Source data are found in Table S1. (E) Western blot analysis on whole-cell lysates of iron markers in WT and LRRK2G2019S macrophages. GAPDH serves as the loading control. Blots represent one of three independent experiments. (F) Levels of ferric (Fe3+) and ferrous (Fe2+) iron in WT and LRRK2G2019S macrophages. Iron is quantified as micromoles of iron per mg of protein. Prior to iron quantification, cells were treated for 16 h with 100 μM ferrous ammonium sulfate (FAS). The mean±s.d. of three biological replicates are shown. (G) Representative images of mCherry–FTL distribution in WT and LRRK2G2019S macrophages, treated without (control, CTRL) or with 250 μM deferoxamine (DFO) for 24 h. Lysosomes were stained with 100 nM LysoTracker (green). Scale bars: 4 μm (left); 1 μm (magnified views). (H) Qualitative classification of mCherry–FTL distribution shown in G. Cells analyzed per biological replicate: WT control, n=80, 83, 74; LRRK2G2019S control, n=109, 94, 97; WT DFO, n=69, 63, 56; LRRK2G2019S DFO, n=84, 78, 79. The mean±s.d. of three separate biological replicates are shown. (I) Western blot analysis of NCOA4, FTL (short and long exposures), FTH and TfR levels on whole-cell lysates of WT and LRRK2G2019S macrophages, treated without (control, ‘C’) or with 250 μM DFO for 24 h. PCNA serves as the loading control. The black arrow indicates the proteolytic cleavage product of FTL. Blots represent one of three independent experiments.
Fig 2: LRRK2G2019S macrophages show defects in NCOA4 turnover upon iron overload. (A) Representative images of mCherry–NCOA4 distribution in WT and LRRK2G2019S macrophages, treated without (control, CTRL) or with 100 μM FAS for 16 h. Lysosomes were stained with an anti-LAMP1 antibody (green). Scale bars: 4 μm (left); 1 μm (magnified views). (B) Quantification of the average size of cytoplasmic mCherry–NCOA4 condensates calculated as μm2 per cell of data shown in A. Cells analyzed per biological replicate: WT control, n=26, 20, 26; LRRK2G2019S control, n=36, 33, 32; WT FAS, n=40, 44, 43; LRRK2G2019S FAS, n=22, 27, 23. The mean±s.d., single values of three biological replicates and P-values calculated using one-way ANOVA with Tukey's post hoc test for multiple comparisons are shown. (C) Quantification of the average number of cytoplasmic mCherry–NCOA4 condensates per cell of data shown in A. For each condition, the number of analyzed cells is the same as that in B. The mean±s.d. and single values of three separate biological replicates and P-values calculated using one-way ANOVA with Tukey's post hoc test for multiple comparisons are shown. (D) Representative time-lapse sequences of mCherry–NCOA4 condensates in WT and LRRK2G2019S macrophages treated for 48 h with 100 μM FAS. Lysosomes were stained with LysoTracker Green (LTG). Time-lapse experiments were acquired for 5 min, capturing both channels every 2 s. Scale bars: 4 μm (left); 1 μm (insets). The video sequence represents events observed consistently across three biological replicates from cells analyzed in B. (E) Western blot analysis of Triton X-100-soluble and -insoluble NCOA4 levels in WT and LRRK2G2019S RAW264.7 macrophages first treated without or with 100 μM FAS for 16 h, and then with DMSO or 25 nM bafilomycin (BAF) for 3 h. GAPDH and TOMM40 serve as the loading controls. Blots represent one of three independent experiments. (F) Western blot analysis of NCOA4 on whole-cell extracts of WT and LRRK2G2019S macrophages left untreated (CTRL) or treated with 100 μM FAS for 16 h, and then not treated (NT) or treated with 1 μM MLi-2 for 8 h in the absence of iron. Phosphorylation levels of LRRK2 on S1292 serve as the positive control, actin serves as the loading control. Blots represent one of three independent experiments.
Supplier Page from DNASU for NCOA4 (Homo sapiens) in pDONR221 (Gateway donor/master vector)