Ix71s1f 3

For cell staining, cultures were washed twice with 1× phosphate-buffered saline (PBS) (Welgene, Republic of Korea) and fixed in 4% formaldehyde (Sigma-Aldrich, Inc., Saint Louis, MO, USA) in PBS for 15 min at room temperature. The cells were then washed thrice with 1× PBS and permeabilized with 0.1% Triton-X-100 (USB Corporation, OH, USA) in PBS for 10 min at room temperature. The cells were washed thrice with 1×PBS and blocked with a blocking solution containing 1% bovine serum albumin (BSA) (Amresco, France), 22.52 mg/ml glycine (Affymetrix, CA, USA), and 0.1% Tween 20 (Affy-metrix, CA, USA) in PBS for 60 mins. Subsequently, the cells were incubated with primary antibodies diluted in dilution solution containing 1% BSA (Amresco, France) and 0.1% Tween 20 (Affymetrix, CA, USA) overnight at 4℃. The cells were then washed thrice with 1× PBS containing 0.1% Tween-20 (PBST) and incubated with secondary antibodies for 2 h at room temperature. Cells were then washed thrice with PBST and incubated with 1 μg/ml DAPI (Sigma-Aldrich, MO, USA) for 5 min at room temperature to stain the nuclei. The samples were subsequen-tly visualized using a fluorescence microscope (IX71S1F3, Olympus, Japan). All antibodies used in this study are listed in Supplementary Table S2.

Calcium imaging was performed on MAP2⁺/NeuN⁺/vGLUT1⁺ neuron-like cells derived from human fibroblast using Rhoda-2-AM (Sigma-Aldrich, Inc., Saint Louis, MO, USA) at a concentration of 2 μM. Imaging was performed in Live-Imaging Solution (Invitrogen, Thermo Fisher Scie-ntific, Inc., Waltham, MA, USA). Images were acquired at 30 frames/s using a scientific CMOS camera. The microscope was controlled by Micro-Manager software and the image processor ImageJ. The samples were visualized using fluorescence microscopy (IX71S1F3, Olympus, Tokyo, Japan). Changes in fluorescence were measured for individual cells, and the average of the first 10 time-lapse images for each region of interest (ROI) was defined as the initial fluorescence (F₀).

K7M2 cells were seeded in 6-well plates. After culturing for 24 h, the medium was replaced with mFeD nanoparticles (1 mg mL⁻¹, 50 μL) and M-mFeD nanoparticles (1 mg mL⁻¹, 50 μL) for 2 and 4 h, respectively. The cells were then washed three times with PBS and fixed with 4% paraformaldehyde solution at room temperature. Finally, the cell nuclei were stained with DAPI for fluorescence microscopy (IX71S1F-3, OLYMPUS, Japan). In addition, flow cytometry was used to quantify the fluorescence intensity. According to the same method above, the K7M2 cells were seeded in 6-well plates, and after the incubation with different nanoparticles, the cells were collected and analyzed using a CytoFLEX flow cytometer (Beckman Coulter, USA).
In addition, SKOV3 cells were seeded in 6-well plates. After culturing for 24 h, the medium was replaced with mFeD nanoparticles (1 mg mL⁻¹, 50 μL) and M-mFeD nanoparticles (1 mg mL⁻¹, 50 μL) for 4 h. Then, the cells were washed three times with PBS and fixed with 4% paraformaldehyde solution at room temperature. Finally, the nuclei were stained with DAPI and the cells were observed under fluorescence microscopy.

HT-1080 cells were seeded in matrigel (BD Biosciences) coated 96-well plate (50 μl per well). After 20-min incubation, the standard medium was replaced by that containing 5% matrigel. Cells were cultured and fluorescence microscopy images were obtained (IX71S1F3, Olympus) on days 1 and 6.