Abc294640

ABC294640 (MedChemExpress) was first dissolved in dimethyl sulfoxide (DMSO) to make a 30 mM stock solution. The working concentration was achieved by adding appropriate amount of stock solution to the bath solution. Vortex was used to facilitate dissolving of ABC294640. Same amount of DMSO was included in the bath solution for the control experiments. In addition, we also tested the effect of DMSO on the channel currents. The highest dose of DMSO (0.2%, v/v) used in our patch-clamp experiments had no effect on the peak and persistent K_V1.4 currents (data not shown). K145 (MedChemExpress) was dissolved in H₂O to make a 10 mM stock solution. S1P (Cayman Chemical) was dissolved in 0.3 M NaOH solution to make a 10 mM stock solution.

All general biochemicals were from Sigma (Poole, UK). ABC294640 was from MedChemExpress (USA). SKi and PF543 were from Merck Biosciences (Nottingham, UK). RB-005 and F-02 were synthesized as described previously [7 (link), 8 ]. MG132 was from Enzo Life Sciences (Exeter, UK). SK2 selective inhibitor (R)-FTY720 methylether (ROMe) was synthesized as described previously [16 (link)]. Antibodies were obtained as follows: anti-actin (#A2066), and anti-p53 (#P8999) from Sigma (Poole, UK); anti-myc (9E10) from Santa Cruz Biotechnology (Santa Cruz, CA), and anti-p21 (#2947) from New England Biolabs (Oxford, UK); anti-DES1 (EPR9680), antibody from Abcam; Anti-SK1 (lab reference number 48:2) antibody was custom made by Abcam using antigens detailed in [28 (link)]. DharmaFECT^™ reagent, ON-TARGETplus SMARTpool^® SK1, SK2 and DES1 siRNAs were from Dharmacon (Cromlington, UK). Scrambled siRNA (ALLSTARS Negative control) was from Qiagen (Crawley, UK).

Female NCI A/JCr mice 4-weeks-old were bought from Charles River Laboratories (Frederick). Neuro-2A cells were subcutaneously implanted in A/J recipient mice by injecting 1 × 10⁶ cells in 100 µl of PBS in the right flank. When Neuro-2A cells formed a palpable tumor (about 6 days), treatments started. The treatments were administrated twice a day for SR59230A and Vehicle, and once a day for ABC294640 and CYM5520. SR59230A (Tocris Bioscence) was delivered at 10 mg/kg of physiological solution via intraperitoneal (i.p.); ABC294640 (MedChem Express) was delivered at 30 mg/kg in 0,375% of Polysorbate 80 in PBS via per os (p.o); CYM5520 (Sigma Aldrich) was delivered at 5 mg/kg in 3.6% DMSO in PBS via i.p. Tumor growth rate was evaluated by measuring tumor mass with a caliber, and tumor mass volume calculated as Volume = [(length × width)²/2]. Mice were sacrificed after 8 days of treatment.