Khanim et al: Supplementary Figure Legends
Supplementary 1: FMC1 Drug library screen of myeloma cell lines
Drugs/agents were dissolved in either DMSO, ethanol or water at 10,000x reported peak serum concentration as observed for routine clinical indications and the library stored at -20’C. Solvent controls were included in the library as internal controls. Prior to use, drugs/agents were diluted in media to a final concentration of 1x peak serum concentration (PSC). Myeloma cells were plated at 2x104 cells in 200ml with 1x PSC drug in triplicate wells in 96 well plates and incubated at 37’C for 72hrs. Viability was measured using CellTiter Blue Assay (Promega) according to manufacturer’s instructions. Background fluorescence from media alone was subtracted from all readings and viability calculated as % of solvent control treated cells. Data is mean± SEM from triplicate experiments
Supplementary Figure 2: Effect of niclosamide dose titration on myeloma cell line viability
Myeloma cell lines were plated in 96 well plates at 2x104 cells/well in 200ml with either solvent control or 1, 2 or 3.2mM niclosamide in triplicate wells. Viable cell number was assessed at 72hours using the CellTitre Blue proliferation assay (Promega) according to manufacturer’s instructions. Background fluorescence from media alone was subtracted from all wells and viability calculated as % of solvent control treated cells. Data shown is the mean ± SEM of triplicate experiments.
Supplementary Figure 3: Effect of niclosamide dose titration and time on primary myeloma sample viability
Total mononuclear cells were purified from primary MM bone marrows aspirates using Ficoll-Paque™ PLUS (GE Healthcare) according to manufacturer’s instructions. Cells were resuspended in RPMI 1640 supplemented with 10% FBS, pen/strep, IL-6, IL-3 and Stem Cell Factor (SCF) at a cell density of 1x106/ml, plated in 24well plates and treated with niclosamide at different concentrations for 24-48hrs. Cell viability was assessed using flow cytometry for CD138/38 and Annexin V FITC/ Propidium Iodide positivity (BD Biosciences). Data shown is viability relative to control for 4 different primary MM samples.
Supplementary Figure 4: Effect of niclosamide on normal donor PBMCs
Total mononuclear cells were purified from the peripheral blood of 4 normal donors using Ficoll-Paque™ PLUS (GE Healthcare) according to manufacturer’s instructions. Cells were resuspended in RPMI 1640 supplemented with 10% FBS, pen/strep at a cell density of 1x106/ml, plated in 24well plates and treated with niclosamide at different concentrations for 24-48hrs. Cell viability was assessed by flow cytometry using viable gates and Cytocount beads. Monocytes, B lymphocytes and T lymphocyte numbers were determined using antibodies to CD14, CD19, CD4 and CD8 respectively followed by flow cytometry. Data shown is as % of total viable cells and is the mean +/- SEM of N=4 normal donors.
Supplementary Figure 5: Characterisation of FLC secretion by MM cell lines
Myeloma cells were washed with media and plated in fresh media at 2.5x105 cells per 200ml in 96 well plates. Supernatant was harvested at 18hrs and analysed for free light chain (FLC) levels using the Freelite assay (Binding Site, UK) and Luminex analysis. Data shown is mean of n=3 experiments +/- SEM.
Supplementary Figure 6: Measurement of respiration rate using Clarks oxygen electrode
Respiration rate was measured using a Clarks oxygen electrode. The electrode chamber was equilibrated with fully air-saturated media alone (1.3ml) and 100% oxygen levels measured. 5x107 cells in 0.2ml media were injected into the electrode chamber using a 19G needle and 1ml hypodermic syringe. Basal respiration was measured until ~60% oxygen remained. To the chamber was then added either 6.7mM FCCP (control uncoupler) or 3.2mM niclosamide. Respiration rate was measured until the electrode reached and equilibrated at 0% O2. Data was recorded on chart paper (A) and the gradients of the lines measured to calculate mmol oxygen turnover per 1x106 cells per minute assuming100% O2=0.257mmol (B).