Fig. 4

Cellular mIBG internalization in IGR-NB8 cells treated with CUDC-907 and several classes of inhibitors. A mIBG internalization in IGR-NB8 cells incubated with DMI, GBR12935 or a combination of both inhibitors with different concentrations (0.01, 0.05, 0.1, 0.5 and 1 μM). Statistical analysis was performed through two-way ANOVA between the respective control (with or without CUDC-907 0.1 μM) and each condition tested. *p < 0.05; **p < 0.01; ****p < 0.0001. Statistical analysis between controls with or without CUDC-907 were performed through the unpaired t-test. ^####p < 0.0001. B mIBG internalization in IGR-NB8 following NET and/or DAT downregulation and CUDC-907 treatment. Statistical analysis was performed through the unpaired t-test using either DMSO (*) or siRNA non-targeting sequence (SiRNA ctrl) (#) as control and comparing with each of the conditions tested. **p < 0.01; ^#p < 0.05. siRNA ctrl is a non-targeting sequence used as a negative control to distinguish specific and non-specific silencing. C and D qPCR quantification of NET and GAPDH mRNA concentration in IGR-NB8 transfected by siRNA. DAT mRNA signal was under the limit of quantification (> 33 PCR cycles for all conditions). Reference genes used were GAPDH and EEIF for NET mRNA quantification and NET and EEIF for GAPDH mRNA quantification. The siRNA GAPDH was used as a positive control to evaluate the transfection and gene downregulation efficiency. E mIBG internalization in IGR-NB8 cells incubated with BGT226 in a dose-dependent manner for 48 h. F mIBG internalization following NET and/or DAT downregulation and BGT226 treatment. Statistical analysis was performed through the unpaired t-test using either DMSO (*) or siRNA ctrl (#) as control and comparing with each of the conditions tested. ***p < 0.001; ^##p < 0.01. G mIBG internalization in IGR-NB8 cells incubated with different PI3K, Akt and mTOR inhibitors for 48 h. Statistical analysis was performed through the unpaired t-test between the control and condition tested. **p < 0.01; ***p < 0.001. All experiments were repeated at least three times in duplicates, normalized by a BCA test and expressed as fold of increase or decrease in mIBG internalization compared with non-treated cells