8 well tissue culture chambers

The F127–Folate coated SPION and F127 coated SPION (50 µg Fe/mL) were inoculated into 8 well tissue culture chambers (Sarstedt, Germany) and incubated for 3 h. Following this, the cells were washed 3 times with PBS and fixed in HCHO (4%) for 30 min. Iron nanoparticles were stained with a freshly mixed equal volume of HCl (4%) and potassium ferrocyanide (4%) for 15 min. After that, cells were rinsed twice with water and incubated with 250 µL Nuclear Fast Red for 5 min. In the final step, cell chambers were rinsed with water, dried, and mounted with cover slips.

MDA-MB-231 cells were grown in 8-well tissue culture chambers on slides (Sarstedt, Nümbrecht, Germany) overnight. On the following day, different chambers were treated with EgKI-1 (2 μM) and control buffer. After 24 hours incubation, supernatants were removed and cells were fixed in ice cold methanol for 10 min followed by two washes with PBS. Cells were then permeabilized with ethanol: acetic acid (2:1) at -20^°C for 5 min. After 3 × 1 min rinses in PBS, normal donkey serum (10%) was applied for 20 min at room temperature (RT). Excess normal serum was decanted and mouse anti-EgKI-1 antibody [16 (link)] and rabbit anti-tubulin antibody (Abcam, Cambridge, UK) diluted 1:80 in PBS were applied for 60 min at RT. Cells were washed with three changes of PBS and Alexa fluor donkey anti-mouse 488 and Alexa fluor donkey anti-rabbit 555 diluted 1:200 in PBS were applied for 30 min at RT. After washing with PBS, cells were stained with DAPI (1:35000 in PBS) for 2–5 min followed by another PBS wash. Prolong fluorescence mount (Thermo Fisher Scientific) was then applied to each slide and a coverslip applied. Cells were visualized under a Zeiss 780-NLO point scanning confocal microscope for the presence of EgKI-1 and tubulin. Fluorescence intensity of tubulin and total cell areas were then measured with ImageJ software [24 (link)] and statistically analyzed with GraphPad Prism version 7.

Cell culture reagents including Dulbecco’s modified Eagle’s medium (DMEM) high glucose, Fetal Bovine Serum (FBS), antibiotic–antimycotic solution (Anti-Anti 100X), and 0.25% trypsin-EDTA solution were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Western blot reagents were acquired from Bio-Rad (Hercules, CA, USA). Antibodies against β-actin, troponin I, and atrial natriuretic peptide were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). The autophagy assay kit was supplied by ABCAM (Cambridge, UK). Annexin-V-FLUOS staining kit was provided by Roche (Nonnenwald, Germany). Annexin-V-FITC was provided by Biolegend (San Diego, CA, USA), and Annexin-V buffer and Propidium Iodine were supplied by BD (New Jersey, USA). Sterile plastic material for tissue culture and 8-well tissue culture chambers were obtained from Sarstedt (Nümbrecht, Germany). MitoTracker Green FM and LysoTracker were from Invitrogen (Carlsbad, CA, USA). ZnO nanopowder < 50 nm particle size (BET), >97% (cat. No. 677450), and all other reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA).