Figure 3.
Ferritin–NCOA4 condensate formation is driven by NCOA4 self-interaction and responsible for ferritin degradation. (A) Truncated constructs of NCOA4. IDR, intrinsically disordered region. (B) WT or the truncated mutants of FLAG-NCOA4 were stably expressed in NCOA4 KO HeLa cells harboring mGFP-FTH1. Scale bars, 10 μm (main) and 1.5 μm (inset). (C) Numbers of mGFP-FTH1 puncta per cell in B were counted (n = 110–151 cells from three biological replicates). Solid bars indicate the means, boxes the interquartile ranges, and whiskers the 10th to 90th percentiles. Differences among the cells expressing FLAG-NCOA4 were statistically analyzed by Dunnett’s multiple comparison test (two-tailed test). Data distribution was assumed to be normal, which was not formally tested. (D) Defective self-interaction of the N-terminal domain mutants of NCOA4. NCOA4 KO HeLa cells expressing both FLAG-NCOA4 (full-length) and mGFP-NCOA4N (the N-terminal domain of NCOA4) with or without I56E or L63R mutation were subjected to coimmunoprecipitation using anti-FLAG antibody. The samples were analyzed by immunoblotting with the antibodies against FLAG, GFP, FTH1, and FTL. (E) The I56E or L63R mutant of FLAG-NCOA4 was stably expressed in NCOA4 KO HeLa cells harboring GFP-FTH1. The cells were observed as in A. (F) Cells used in B were grown in DMEM and treated with 10 μg/ml FAC for 24 h followed by 50 μM DFO for 12 h. Whole-cell lysates were analyzed by immunoblotting with the antibodies against FTH1, FTL, HSP90, and FLAG. Asterisk represents a putative degradation product of FTH1. (G) FTH1 degradation upon DFO treatment in F. The ratio of the endogenous FTH1 band intensities under DFO-treated conditions to those under FAC-treated conditions is shown. Solid bars indicate the means, and dots the data from three independent experiments. Differences were statistically analyzed by Dunnett’s multiple comparison test (two-tailed test). Data distribution was assumed to be normal, which was not formally tested. (H) The cells used in E were examined as in F. (I) Quantification of H as in G. Source data are available for this figure: SourceData F3.

Ferritin–NCOA4 condensate formation is driven by NCOA4 self-interaction and responsible for ferritin degradation. (A) Truncated constructs of NCOA4. IDR, intrinsically disordered region. (B) WT or the truncated mutants of FLAG-NCOA4 were stably expressed in NCOA4 KO HeLa cells harboring mGFP-FTH1. Scale bars, 10 μm (main) and 1.5 μm (inset). (C) Numbers of mGFP-FTH1 puncta per cell in B were counted (n = 110–151 cells from three biological replicates). Solid bars indicate the means, boxes the interquartile ranges, and whiskers the 10th to 90th percentiles. Differences among the cells expressing FLAG-NCOA4 were statistically analyzed by Dunnett’s multiple comparison test (two-tailed test). Data distribution was assumed to be normal, which was not formally tested. (D) Defective self-interaction of the N-terminal domain mutants of NCOA4. NCOA4 KO HeLa cells expressing both FLAG-NCOA4 (full-length) and mGFP-NCOA4N (the N-terminal domain of NCOA4) with or without I56E or L63R mutation were subjected to coimmunoprecipitation using anti-FLAG antibody. The samples were analyzed by immunoblotting with the antibodies against FLAG, GFP, FTH1, and FTL. (E) The I56E or L63R mutant of FLAG-NCOA4 was stably expressed in NCOA4 KO HeLa cells harboring GFP-FTH1. The cells were observed as in A. (F) Cells used in B were grown in DMEM and treated with 10 μg/ml FAC for 24 h followed by 50 μM DFO for 12 h. Whole-cell lysates were analyzed by immunoblotting with the antibodies against FTH1, FTL, HSP90, and FLAG. Asterisk represents a putative degradation product of FTH1. (G) FTH1 degradation upon DFO treatment in F. The ratio of the endogenous FTH1 band intensities under DFO-treated conditions to those under FAC-treated conditions is shown. Solid bars indicate the means, and dots the data from three independent experiments. Differences were statistically analyzed by Dunnett’s multiple comparison test (two-tailed test). Data distribution was assumed to be normal, which was not formally tested. (H) The cells used in E were examined as in F. (I) Quantification of H as in G. Source data are available for this figure: SourceData F3.

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