Sbi 0206965

Soleus and extensor digitorum longus (EDL) muscles were excised from anesthetized mice (6 mg pentobarbital and lidocaine/100 g body weight, intraperitoneal injection) and suspended in incubation chambers (Multi Myograph System, Danish Myo-Technology, Hinnerup, Denmark) containing 30 °C Krebs Ringer Henseleit (KRH) buffer supplemented with 8 mM mannitol and 2 mM pyruvate as previously described [22 (link)]. The muscles were pre-incubated for 20–40 min with the ULK1/2 inhibitor, SBI-0206965 (#18477, Cayman Chemical Company, Ann Arbor, MI, USA) or DMSO (Sigma) as a control, and subsequently stimulated with either 60 nM insulin (Actrapid, Novo Nordisk) for 20 min or 4 mM 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR (Toronto Research Chemicals, North York, ON, Canada)) for 40 min with or without the inhibitors present. The total duration of the incubation experiments was 1 h.

All cell lines were cultured in DMEM with 10% FBS unless otherwise noted. Stable TFEB-KO line was generated using gRNA in Lenticrispr V2 (Addgene plasmid 49535) (63 (link)) using the guides listed (Supplemental Table 3). Constructs for doxycycline-inducible shRNA were generated using the Tet-pLKO-puro (Addgene plasmid 21915). Plasmids were generated and inserted in to a lenti-viral vector for stable transfection. Knockdown was induced using 200 ng/mL of doxycycline for 48 hours. The HCT116 cells used for tracking mitophagy were generated from the pCLBW Cox8-mCherry-EGFP plasmid (Addgene plasmid 78520). ATG4B mutant–expressing cell line was developed by stable expression of pmStrawberry-Atg4B^C74A (Addgene plasmid 21076). We generated the HCT116 LysoIP line using the pLJC5-Tmem192-3xHA (Addgene plasmid 102930). Cells were treated chloroquine diphosphate (MilliporeSigma) and SBI-0206965 (Cayman Chemical) using concentration and time as shown in the figure.

GFP-AZI2 -4OHT cells were seeded in 24-well plates and left overnight before treatment with respective inhibitors. Upon treatment, images of cells were acquired every 2 h with an Incucyte live-cell imaging system (Essen Bioscience) to monitor GFP-AZI2 puncta formation. Inhibitor concentrations used in this screen were; SB203580, 10 µM (MedChemExpress, HY-10256); Tozasertib, 5 µM (MedChemExpress, HY-10161); Chloroquine, 100 µM (Sigma Aldrich, C6628); GDC0994, 5 µM (Cayman Chemical, 21107); Prexasertib, 5 µM (MedChemExpress, HY-18174); LTURM34, 10 µM (MedChemExpress, HY-101667); GSK650394, 5 uM (MedChemExpress, HY-15192); Amlexanox, 100 µM (MedChemExpress, HY-B0713); U0126, 10 µM (Cayman Chemical, 70970); IRAK 1–4 Inhibitor I, 10 µM (MedChemExpress, HY-13329); SP600125, 25 µM (MedChemExpress, HY-12041); SBI0206965, 10 µM (Cayman Chemical, 18477); BMS345541, 10 µM (MedChemExpress, HY-10519); Triciribine, 10 µM (MedChemExpress, HY-15457); Silmitasertib, 10 µM (MedChemExpress, HY-50855); PP242, 5 µM (MedChemExpress, HY-10474); Lys05, 20 µM (provided by Dr. Ravi Amaravadi); Palbociclib, 5 µM (MedChemExpress, HY-50767); and D4476, 50 µM (MedChemExpress, HY-10324). The puncta/image at 24 h were quantified and plotted.

R481 and R419 were kindly gifted by Rigel Pharmaceuticals Inc (San Francisco, USA). Chemical structures for compounds are shown in Figure 1. SBI-0206965 was purchased from Cayman Chemical, hexamethonium bromide from Sigma Aldrich, 50% glucose solution from Centaur Services, and Novo Nordisk Actrapid insulin was purchased from Covetrus.