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. 2022 Mar 23;23(5):e54278. doi: 10.15252/embr.202154278

Figure 2. Multivalent interactions are required for NCOA4 condensation under iron‐replete conditions.

Figure 2

  1. Schematic diagram of NCOA4 variants used in this study.
  2. NCOA4 KO MEFs reconstituted with NCOA4 variants were treated or not treated with 10 μg/ml FAC for 12 h and then fixed for imaging. Cells were immunostained with an anti‐myc antibody. Representative images are shown. Scale bar, 10 μm.
  3. Number of puncta per cell in (B) is shown as the means ± SEM of at least 40 cells in each condition from two biological replicates.
  4. NCOA4 KO MEFs reconstituted with NCOA4 variants were treated or not treated with 10 μg/ml FAC for 12 h, fractionated, and analyzed by immunoblotting with the indicated antibodies.
  5. DISOPRED3 disorder score of human NCOA4.
  6. Wild type MEFs stably expressing NCOA4 IDR (aa 167–334) fused with tandem FKBP were treated with 10 μg/ml FAC or 20 μM Dfo for 12 h and then cultured with 0.1 nM FKBP ligand (AP20187) for 6 h. The cells were fixed for imaging and immunostained with anti‐myc to analyze the puncta of the fusion protein. Scale bar, 10 μm.
  7. Number of puncta in (F) are shown as means ± SEM of at least 35 cells in each condition from two biological replicates. P < 0.0001 (Dfo + ligand vs F.AC + ligand) and P = 0.048 (FAC vs. FAC + ligand; Kruskal–Wallis ANOVA with Dunn's multiple comparison test). P values were adjusted using the Bonferroni method.
  8. Coomassie‐stained SDS–PAGE gel of purified proteins.
  9. The amount of co‐purifying iron in purified proteins in (H) was measured by inductively coupled plasma mass spectrometry (ICP–MS). Data are shown as means ± SD of three biological replicates. P = 0.032 (Welch two sample t‐test).

Source data are available online for this figure.