Fig. 1: H-ferritin uptake by human macrophages is an endocytosis-dependent process.

a Flow cytometry analysis of internalized HFt-AF488 by primary CD4⁺ and CD8⁺ lymphocytes, monocytes and macrophages within 30 min at 37 °C; n = 3 biologically independent replicates. b Flow cytometry analysis of internalized HFt-AF488 by primary monocytes and hMDM at indicated concentrations and time points at 37 °C; n = 3 biologically independent replicates. c Flow cytometry analysis of internalized HFt-AF488 by hMDM at various concentrations (5, 10, 25, 50, 100, 500 μg/ml) within 30 min at 4 or 37 °C; n = 3 biologically independent replicates. d Representative confocal microscopy images of internalized HFt-AF488 (green) by hMDM at a concentration of 50 μg/ml within 30 min at 4 or 37 °C. After HFt-AF488 uptake, cells were fixed and stained with anti-EEA1 antibody (red) and Hoechst 33342 (blue). Merged fluorescence images show colocalization of EEA1 marker and HFt-AF488 (yellow foci) in macrophages incubated at 37 °C. Scale bar = 20 μm. e Flow cytometry analysis of internalized HFt-AF488 (100 μg/ml) by hMDM at indicated time points at 37 °C; n = 3 biologically independent replicates. f Representative confocal microscopy images of internalized HFt-AF488 (green) by hMDM at a concentration of 50 μg/ml within 60 min at 37 °C. After HFt-AF488 uptake, cells were fixed and stained with anti-LAMP1 antibody (red) and Hoechst 33342 (blue). Merged fluorescence images show colocalization of LAMP1 and HFt-AF488 (yellow foci) in macrophages. Scale bar = 20 μm. g Flow cytometry analysis of internalized HFt-AF488 (10 μg/ml) by hMDM in the absence or presence of indicated concentrations of unlabeled HFt within 30 min at 37 °C; n = 3 biologically independent replicates. h Representative confocal microscopy images of internalized HFt-AF488 (10 μg/ml) (green) by hMDM in the absence or presence of unlabeled HFt (2.5 mg/ml) within 30 min at 37 °C. After HFt-AF488 uptake, cells were fixed and stained with anti-EEA1 antibody (red) and Hoechst 33342 (blue). Scale bar = 20 μm. i–k Flow cytometry analysis of internalized HFt-AF488 (50 μg/ml) by hMDM in the absence or presence of various inhibitors of macropinocytosis: EIPA (i), rottlerin (j) and cytochalasin D (k) at indicated concentrations within 30 min at 37 °C; n = 3 biologically independent replicates. l Representative confocal microscopy images of internalized HFt-AF488 (50 μg/ml) (green) by hMDM in the absence or presence of cytochalasin D (10 μM) within 30 min at 37 °C. After HFt-AF488 uptake, cells were fixed and stained with Hoechst 33342 (blue). Scale bar = 20 μm. Flow cytometry data are presented as the mean fluorescence intensity (MFI) of HFt-AF488. Data are presented as mean ± SEM. The two-way ANOVA and Tukey’s post-hoc tests were used for statistical analysis in panels a and b. The one-way ANOVA and Dunnett’s post-hoc tests were used for statistical analysis in panels g and i-k. For all panels, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Source data are provided as a Source Data file.