mIBG internalization in cell lines

In all experiments, cells were seeded in 48-well plates and grown at 37 °C for 24 h. mIBG internalization was blocked by rapid removal of the medium containing mIBG and by adding cold PBS for three washes before lysing the cells with 100 µL 0.2% Tween-20 in PBS. Intracellular and extracellular mIBG were then extracted and quantified. Concentrations were normalized to the total protein content in each sample using the bicinchoninic acid (BCA) protein assay (ThermoScientific).

mIBG was added for 0, 1 and 4 h at a concentration of 100 nM.

HEK-transfected cells were incubated with mIBG at different concentrations (from 0 to 20 µM) for 10 min at 37 °C.

Specificity of DMI and {"type":"entrez-protein","attrs":{"text":"GBR12935","term_id":"2281712181","term_text":"GBR12935"}}GBR12935 toward NET and DAT, respectively, was determined by increasing concentrations of inhibitors (from 0 to 10 µM) at 37 °C, 30 min before mIBG incubation. To study the effect of NET and DAT inhibition on mIBG internalization in CUDC-907-treated cells, cells were incubated with CUDC-907 0.1 µM for 48 h following DMI and/or {"type":"entrez-protein","attrs":{"text":"GBR12935","term_id":"2281712181","term_text":"GBR12935"}}GBR12935 treatment at different concentrations (from 0 to 1 µM) for 30 min prior to mIBG incubation (10 nM for 10 min). For testing HDAC and PI3K/AkT/mTOR inhibition effects, cells were treated for 48 h at 37 °C and incubated with mIBG 10 nM for 10 min.

IGR-NB8 cells were treated with CUDC-907 for 48 h and mIBG 10 nM was added in the different wells for 10 min. Cells were then washed with pre-warmed PBS and finally culture medium with or without CUDC-907 0.1 µM was added to the cells for a further incubation time corresponding to 5 min, 1, 6, 12, 24 and 48 h.