Thp 1 monocytic cell line

In order to validate the previous RT-qPCR results, the protein expression level of RAGE was verified by Western blot. Cells were cultured in T-75 flasks and treated under the same experimental conditions mentioned above. As a positive control of constitutive RAGE expression, THP-1 monocytic cell line (ATCC, Manassas, VA, USA) was used (Supplementary Figure S1). Following treatment, cytoplasmic and cell membrane extracts were obtained using the Subcellular Protein Fractionation Kit for Cultured Cells (Thermo Scientific, Waltham, MA, USA). Protein was quantified using the MicroBCA Protein Assay Kit (Pierce, Waltham, MA, USA). A total of 30 mg of protein per lane was resolved in 10% SDS-PAGE and subsequently transferred onto a nitrocellulose membrane. After 1 h of blockade of non-specific sites with 2% of Bovine Serum Albumin in PBS, membranes were incubated with primary antibodies against RAGE (Abcam, Cambridge, MA, USA) or β-Actin (Sigma, Burlington, MA, USA) (1:200) at 4°C overnight. After washing, the membranes were incubated with biotinylated anti-rabbit (Dako, Santa Clara, CA, USA) or anti-mouse (GeneTex, Alton Pkwy Irvine, CA, USA) secondary antibodies (1:300) for 1 h at RT. Protein bands were visualized by employing the Vectastain Elite ABC peroxidase kit (Vector Laboratories, Burlingame, CA, USA) and DAB-H₂O₂ system (Sigma, Burlington, MA, USA).

Cell culture. Human THP-1 monocytic cell line was obtained from American Type Culture Collection (ATCC, TIB-202TM, Manassas, VA, USA). Cells were grown at 37 °C under 5% CO₂ atmosphere in RPMI 1640 medium supplemented with 10% of heat-inactivated fetal bovine serum, 100 U/mL of penicillin, 100 µg/mL of streptomycin and 0.25 µg/mL of amphotericin. They were split every three days.
Cell viability. Cell viability assay was analysed using the WST-1 assay (Roche, 11644807001, Meylan, France), according to manufacturer’s protocol [31 ]. THP-1 cells were seeded at 5 × 10⁴ cells/mL in 96-well plates and exposed to different concentrations of MIP-capped CdTe_0.5S_0.5/ZnS QDs. After 24 h of exposure, WST-1 reagent was added in each well. Cells were incubated at 37 °C for 2 h. The absorbance of the solution was determined at 480 nm on microreader (BioRad-iMARK, Marnes la Coquette, France) to calculate the IC₅₀ for each QD. Each experiment is carried out on three independent biological replicates.

A THP‐1 monocytic cell line (American Type Culture Collection, Manassas, VA) were maintained as per ATCC guidelines in culture medium and 0.05 mmol/L ß‐mercaptoethanol. was differentiated into macrophages by seeding at a density of 0.75 × 10⁵ cells in 48 well plastic plates or 1.5 × 10⁵ into 24 well plates, then stimulating with 45 μmol/L phorbol 12‐myristate 13‐acetate (PMA) for 72 h as previously described (Ween et al. 2016). THP‐1 monocytes were differentiated three days prior to treatment. Experiments were carried out within 10 passages and THP‐1 cells were discarded at passage 30 to avoid contamination and genetic mutations from the original source.

The THP-1 monocytic cell line was obtained from American Type Culture Collection (ATCC, Rockville, MD, USA) and cultured in RPMI 1640 medium (Invitrogen, Waltham, CA, USA), supplemented with 10% fetal bovine serum (Invitrogen) and 1% penicillin-streptomycin (Invitrogen). The cells were sub-cultured twice a week. THP-1 cells were differentiated into macrophages by incubating with phorbol 12-myristate 13-acetate (50 µM for 24 h). Differentiated macrophages were used for experiments after incubation in normal media for two days. In the experiments, the cells were exposed to 100 µg/mL ferric ammonium citrate (FeAC) for 24 h or pretreated with 1 mM iron chelator deferoxamine (DFO) for 1 h and then exposed to FeAC for 24 h without DFO in the culture media. Untreated cells were used as controls.