Ic261

H-89 dihydrochloride, D4476, IC261, LY-364947, DAPH 2, nafoxidine hydrochloride, 1,3-di-o-tolylguanidine, naftifine hydrochloride, opipramol, rifampicin, kanamycin, and the library of pharmacologically active compounds (LOPAC) were purchased from Sigma-Aldrich, Zwijndrecht, the Netherlands. Hygromycin B was acquired from Life Technologies-Invitrogen, Bleiswijk, the Netherlands. Imatinib mesylate was from Enzo Life Sciences, Raamsdonksveer, the Netherlands. VEGFR2 kinase inhibitor I and ampicillin were purchased from Calbiochem Merck-Millipore, Darmstadt, Germany. Pazopanib HCl, AT9283, and linifanib (ABT-869) were acquired from Selleck Chemicals, Munich, Germany. Quizartinib was purchased from MedChemExpress, Stockholm, Sweden. Santa Cruz BioTechnology, Heidelberg, Germany was the supplier of PDGFR tyrosine kinase inhibitor III. Dovitinib (TKI-258, CHIR-258) was from APExBIO, Houston, TX, USA. The siKinome library was acquired from Thermo Fisher Dharmacon, Waltham, MA, USA.

THP-1 cell lines were cultured in RPMI supplemented with 10% FCS, 100 μg ml⁻¹ of penicillin and 100 μg ml⁻¹ of streptomycin and 1 μg ml⁻¹ puromycin. ABT-199 was purchased from ChemieTek (Indianopolis, IN). Cantharidin, digoxigenin, proscardillin, wortmannin, SB203580, TOFA, IC 261 and vorinostat were all purchased from Sigma. ABT-737, GSK-J4, GSK-126, G007-LK, MM-102, SKI-606 and JNK-IN-8 were purchased from Sellekchem (Houston, TX). LB100 (HY-18597) was obtained from MedChemExpress (MonMouth Junction, NJ). After 4 days of Dox induction (or control without Dox), cells were plated in RPMI 20% foetal bovine serum at 2 × 10⁵ ml⁻¹ in 96-well plate with twofold dilutions of each drug performed in duplicate. At 72 h, cell viability was measured using a plate-reader after addition of 10% Presto Blue Cell Viability Reagent (ThermoFisher Scientific) at emission fluorescence 590 nm. IC₅₀ curves were calculated for each drug in the presence and absence of Dox using GraphPad Prism 6.0 (dose response inhibition) and the difference in IC₅₀ was plotted as a percentage of control (no dox). For ACACA validation, transduced THP-1 cells were induced with Dox for 3 days, then washed into low serum RPMI (0.5%) and cultured at low cell density for 7 days +/−TOFA before cell growth was measured with Presto Blue on a plate reader.

LRRK2 kinase inhibitors CZC-25146 and PF-06447475 and casein kinase 1 inhibitor IC261 were purchased from Sigma-Aldrich. Casein kinase 1 inhibitor PF-670462 was purchased from Abcam. LRRK2 kinase inhibitor MLi-2 was kindly provided by Dr. D. Alessi (Division of Signal Transduction Therapy, University of Dundee). pCHMWS_3Flag_LRRK2_Ires_Hygro constructs of pathogenic (R1441C/G, Y1699C, and I2020T) LRRK2 variants were cloned as described in [42 (link)]. The pCHMWS_3Flag_LRRK2_Ires_Hygro constructs encoding truncated variants PLRCKW and APLRCKW, as well as the LRRK2 S908A/S910A/S935A/S955A/S973A/S976A or S908E/S910E/S935E/S955E/S973E/S976E were generated using gBlock® Gene Fragments (IDT) and as described in [42 (link)]. The following antibodies were used: mouse anti-FlagM2 (Sigma-Aldrich, F1804), mouse anti-vinculin (Sigma-Aldrich, V9131), mouse anti-α-tubulin (Sigma-Aldrich, T5168), rabbit anti-LRRK2 P-S935 (Abcam, ab133450), rabbit anti-LRRK2 P-S1292 (Abcam, ab203181), rabbit anti-LRRK2 MJFF-2 antibody (Abcam, ab133474), mouse anti-LRRK2/Dardarin, N-terminus N138/6 (Neuromab 75-188), mouse anti-LRRK2/Dardarin, C-terminus N241A/34 (Neuromab 75-253), mouse anti-LRRK2 MC.028.83.76.242 (ab130277).