Methyl thiazolyl tetrazolium (mtt)

Cell viability was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay. A549 cells were first incubated in each well of a 96-well plate (Corning Inc., Corning, NY) for a total of 24 hours. Ferrofluids of varying concentrations (0.05%, 0.19%, 0.22%, and 0.26% v/v) were added to the plate. After incubation for 12 hours, ferrofluid and medium were removed and cells were washed three times with PBS. MTT (ATCC, Manassas, MA) assay was then performed to determine the cell viability following the manufacture’s recommended protocol. Cell viability of the other 6 cell lines was investigated by the same MTT assay with a 0.26% (v/v) ferrofluid after 2 hours’ incubation.
Cell proliferation rate was assessed by MTT assay, too. A549 cells were first incubated with ferrofluids (0.26% v/v) for 1 minute and 2 hours, respectively at 37 °C under a humidified atmosphere of 5% CO₂. Cells were then washed three times with PBS and released through incubation with 0.05% Trypsin-EDTA solution. 4000 cells were seeded in each well of a 96-well plate. MTT assay was performed every 24 hours to determine the growth rate following the manufacture’s recommended protocol. The medium was changed on the third day. The proliferation of other 6 cancer cell lines was investigated by attempting to culture cells to confluence after exposing them to ferrofluids for 2 hours.

A broth microdilution method was used to determine the growth inhibition ED₅₀ values. Briefly, ~ 10⁴ NCI-H460 cells suspended in 100 μL of DMEM supplemented with 10% fetal bovine serum (FBS), 4.5 g/L glucose and L-glutamine and preserved with 1% penicillin–streptomycin were seeded in 96-well plates (Corning Inc., Corning, NY) and incubated at 37 °C in a 5% CO₂ atmosphere. The cells were cultured for 24 h then incubated with different concentrations of compounds for another 24 h. Then, an MTT ((3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) cell proliferation assay (ATCC, Manassas, VA) was performed. Data from 4 experiments for each inhibitor were pooled and then fitted to single dose-response curves. The reported values are thus +/− SEM (n=4).

The human tumor cell line NCI-H460 (non-small cell lung cancer) was obtained from the National Cancer Institute and maintained at 100% humidity and 5% CO₂ at 37 °C. Cell growth inhibition assays were carried out as described previously.^{20 (link)} A broth microdilution method was used to determine the growth inhibition IC₅₀ values. Briefly, ~5 × 10⁴ cells suspended in 100 μL of DMEM supplemented with 10% fetal bovine serum (FBS), 4.5 g/L glucose and L-glutamine and preserved with 1% penicillin–streptomycin were seeded in 96-well plates (Corning Inc., Corning, NY) and incubated at 37 °C in a 5% CO₂ atmosphere. The cells were incubated with 1000 μM, 333 μM, 111 μM, 37 μM, 12 μM, 4.1 μM, 1.4 μM, 0.46 μM, 0.15 μM, and 0.051 μM of compounds and H₂O as a control for 4 days.^{20 (link)} Then, an MTT ((3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) cell proliferation assay (ATCC, Manassas, VA) was performed to obtain dose–response curves. The IC₅₀ values of the free BP, Mo₆Zol₂ and Mo₄Zol₂Mn(III), which had been measured in MCF-7 cells in our first studies^{21 (link),22 (link)} were re-determined here using the same conditions as for the other compounds (Table 1).

Antibodies against the following proteins were used in this study: DOK3, C-Cbl, and Grb2 were from Santa Cruz Biotechnology (Santa Cruz, CA, USA); Phospho-ERK (pThr²⁰²/pTry²⁰⁴), ERK (pan ERK), Phospho-p38 MAPK (Thr180/Tyr182), p38 MAPK (pan p38 MAPK), Phospho-Syk (Tyr525/526), Syk (panSyk), IκBα, cyclin D1 antibody were from Cell Signaling Technology (Danvers, MA, USA) ; Anti-phosphotyrosine monoclonal antibody G410 was purchased from EMD Millipore (Billerica, MA, USA). TRAP staining kit was purchased from Sigma (St. Louis, MO, USA). Phospho-Dap12 rabbit antibody was previously described (6 (link)). Murine M-CSF and RANKL were obtained from Peprotech (Rocky Hill, NJ, USA). The enhanced chemiluminescence (ECL) was purchased from Thermo Scientific (Rockford, IL, USA). Tissue culture media and fetal bovine serum (FBS) were obtained from Life Technologies (Grand Island, NY, USA). MTT (3-(4, 5-dimethylthiazole-2-yl)-2, 5-dipenyltetrazolium bromide) cell proliferation assay kit was obtained from ATCC (Manassas, VA, USA).