Cell culture. Hepatic carcinoma HepG2 cells were grown at 37°C in a 5% CO2 humidified environment in Dulbecco’s modified Eagle’s medium (Gibco) supplemented with 10% fetal bovine serum (Biological Industries), penicillin (100 U/ml), and streptomycin (100 μg/ml; HyClone). Cells were treated with FAC (200 μg/ml; F5879-100G, Sigma) for 3 days to induce iron-replete conditions. Before being collected, cells were washed with phosphate-buffered saline (PBS) five times.

Immunoblotting assay. HepG2 cells grown in six-well plates were treated with or without FAC. After washing with PBS, whole-cell lysates were prepared in lysis buffer [20 mM tris-HCl (pH 8.0), 137 mM NaCl, 2 mM EDTA, 1% NP-40, and 1× protease inhibitor cocktail], centrifuged to remove the precipitation, and mixed with SDS–polyacrylamide gel electrophoresis sample loading buffer. Immunoblots were stained sequentially with mouse anti-ferritin light chain (Santa Cruz Biotechnology, sc-74513, 1:800 dilution) and mouse anti–β-actin (Sigma, clone AC-74, 1:2000 dilution) monoclonal antibodies, and with horseradish peroxidase–labeled goat anti-mouse secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA).

Immunofluorescence microscopy. For immunostaining, cells were fixed in −20°C precooled 100% methanol for 5 min and washed with PBS five times at room temperature, each for 5 min. Subsequently, cell samples were blocked in blocking solution [2% bovine serum albumin (BSA), 1× PBS, 0.1% Triton X-100, and 0.05% Na azide] for 30 min at room temperature. Mouse anti-ferritin light chain monoclonal antibody (Santa Cruz Biotechnology, sc-74513) was used at a 1:200 dilution in incubation buffer (1% BSA, 1× PBS, 0.1% Triton X-100, and 0.05% Na azide) overnight at 4°C. Alexa Fluor 488 AffiniPure Goat Anti-Mouse IgG (H+L) (Jackson ImmunoResearch, 115-545-003) was used at a dilution of 1:600 and incubated for 2 hours at room temperature. After extensive washing and 4′,6-diamidino-2-phenylindole (DAPI) staining, the cells were used for confocal imaging. Multiwavelength fluorescence images were acquired using a FLUOVIEW FV1000 microscope (Olympus, Tokyo, Japan) equipped with a 100× NA 1.45 oil-immersion objective and captured using FV10-ASW version 3.1 software. Images were analyzed, and fluorescence intensities were quantified in ImageJ 1.45m (W. Rasband, National Institutes of Health).

Embedded cell sample preparation and TEM imaging. The resin-embedded cell samples were prepared by high-pressure freezing and freeze substitution. The treated cells were instantaneously frozen in a BAL-TEC HPM 010 high-pressure freezer (BAL-TEC). The freeze substitution was performed in an automatic freeze-substitution unit (Leica, AFS 2), and 1% osmium tetroxide (OsO4) was used to fix the cells and quench the autofluorescence in cells. As a result, the fluorescence photon count decreased from several million to several thousand per second.

In the following infiltration and polymerization, the LR White embedding medium (EMS) was chosen for its low fluorescence and EPR signals (fig. S4). The resin-embedded cells were polymerized for 48 hours at 60°C. For using other polymerization processes or some other resins, such as HM20, KM40, and GMA, either the polymerized resins showed strong autofluorescence at 532-nm laser illumination or the polymerization using 365-nm ultraviolet irradiation generated many unknown radicals in the sample, which appeared strong in the EPR spectroscopy. The ultrathin sections (70 to 100 nm) were examined in an HT-7700 TEM system equipped with an AMT (Advanced Microscopy Techniques) CCD camera operated at 80 kV.

For MI imaging, the polymerized cell sample was trimmed with blade and fixed at the end of the AFM-used tuning fork for sectioning. To minimally disturb the property of the tuning fork, the size of the quadrate sample was limited to less than 1 mm × 1 mm × 1 mm. On an ultramicrotome (Leica EM UC7), the sample tip was exquisitely trimmed to a trapezoidal frame by a glass knife, with a size of 50 μm × 50 μm at the bottom and 10 μm × 10 μm at the top and with a height of 50 μm for high-resolution MI in Fig. 4. Then, the surface of the 10 μm × 10 μm cross section was sectioned by a diamond knife (DiATOME) to form a smooth surface with a flatness of a few nanometers, examined by the AFM (fig. S5). Last, we verified that there were one or more cells in the exposed surfaces on the light microscope. For correlated imaging, the tips of the cell samples were trimmed to cubes with a size of 50 μm × 50 μm × 50 μm, whose size was big enough to ensure that we could be able to collect the sections. The last piece of ultrathin sections (100 nm) was collected for the TEM examination.

EPR and mass spectrometric assays. The ensemble EPR was carried out at room temperature on a JES-FA200 spectrometer. The EPR results of liquid samples and embedded samples are shown in Fig. 2 and fig. S4, respectively. Unlike the in vitro measurement with purified ferritin, there was a chance that some other paramagnetic species contributed the spin noise signal, such as manganese (Mn). We carried out mass spectrometric detection to preclude the influence of other possible paramagnetic metal ions. First, cells were washed for five times with PBS and collected. Then, 200 μl of concentrated HNO3 was added into the cells and heated for 2 hours at 90°C. The final cell lysates were diluted with ddH2O to 10 ml. The mass spectra were measured via inductively coupled plasma mass spectrometry. The mass spectra showed a significant increase of the iron concentration in iron-loaded cells compared with that in control cells (0.484 μg/ml versus 0.042 μg/ml). However, there was nearly no detectable Mn in both groups (0.005 and 0.003 μg/ml).

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