Ci 994

The PKIS2 Library was supplied by The Structural Genomics Consortium at the University of North Carolina at Chapel Hill. The PKIS2 library was prepared at 1 mM concentration in DMSO in 384-Well Low Dead Volume (LDV) source plates and were dispensed using the ECHO 550 (Labcyte; San Jose, CA, USA) into a total well volume of 40 µL. The primary screen was performed at 1 µM for 24 h, in duplicate for lysosomal assays (on duplicate plates) and as single replicates for immunocytochemistry for TFEB or TFE3. Each concentration on a dose response was performed in triplicate between 0.01 and 2 µM for 24 h. Additional analogs and commercially available compounds were assayed up to 5 and 20 µM respectively. The control compounds selected for use with the assays were the class I HDAC inhibitor CI-994 (Tacedinaline; Tocris Bioscience, 2952) at 10 µM and the lysosomal inhibitor chloroquine at 20 µM (Sigma-Aldrich, C6628).

2-Deoxy-d-glucose (2-DG, Sigma D6134-1G); NMDA (Tocris, 0114); D-AP5 (Tocris, 0106); MK801 (Tocris, 0924); CGP (Tocris, 1493); DIDS (Sigma, D3514); Veratridine (Tocris, 2918); Bumetamide (Sigma, B3023); GlyH-101 (Sigma, 219671); NPPB (Tocris, 0593); Mannitol (Spectrum Chemical, MA165); Ionomycin (Tocris, 1704); SMIFH2 (Sigma, S4826-5MG); CK-666 (Sigma, SML_0006_5MG); Latrunculin A (Lat-A, Sigma, L5163); Thapsigargin (Tocris, 1138); Jasplakinolide (JASP, Tocris, 2792); Bovine Serum Albumin (BSA, Sigma, A4919-5G); DAPI (Biotium, 40043); SAHA (Tocris, 4652); Santacruzamate A (Tocris, 7191); CI994 (Tocris, 2952); FK228 (Tocris, 3515); Tubastatin (Tocris, 6270); Nicotinamide (Tocris, 4106); EX527 (Tocris, 2780); anti-sirtuin 5 inhibitor peptides (Dr.C.A. Olsen, NRE139; NRD167); Suramin (Fisher, AC328540500); C646 (Sigma, SML0002); Cycloheximide (Sigma, C4859); Blebbistatin (Sigma, B0560); Taxol (Tocris, 1097); Nocodazole, (Tocris, 1228); MyoVin-1, (Calbiochem, 475984); Mibefradil, (Tocris, 2198); ω-Conotoxin GVIA, (Tocris, 1085); NBQX (Tocris, 0373); Nifedipine, (Tocris, 1075). Each chemical was dissolved according to the manufacturer’s guidelines, either in water or DMSO, or ethanol.