Torin1 cay10997

Primary BMDMs and iBMDMs were seeded in 24-, 12-, or six-well plates at a density of 0.5 × 10⁶, 1 × 10⁶, or 2 × 10⁶ cells per well, respectively. Experiments with U18666a (662015; EMD Millipore) were optimized in different cell types so as to block lysosomal efflux of cholesterol and achieve absolute lysosomal accumulation. Primary BMDMs exposed to U18666a displayed lysosomal cholesterol accumulation at 48 h, while in iBMDMs, sufficient cholesterol accumulation was observed by 24 h (Fig. S1). Where used, cells were exposed to Torin1 (CAY10997; Cayman Chemical) for 3 h, and Baf A1 (Sigma-Aldrich) and CQ (Sigma-Aldrich) at the indicated concentrations for 1 h before ATP stimulation.

The LRRK2in1 compound and the VPS34in1 were purchased from the Division of Signal Transduction Therapy, School of Life Sciences, and University of Dundee, UK. The GSK2578215A compound was purchased from Tocris. Bafilomycin A1 (B1793-2UG), Chloroquine (C6628) and Wortmannin (W3144-250UL) were purchased from Sigma-Aldrich. Torin-1 (CAY10997) was purchased from Cayman Chemicals.
Antibodies used were as follows: LC3 antibody (NB100-2220, Novus Biologicals); LRRK2 antibodies (MJFF#2, 3514-1/ab133474, Epitomics); total S6 antibody (2317, Cell Signalling); phospho Ser235/236 S6 antibody (2211S, Cell Signalling); total P70S6K antibody (sc-8418, Santa Cruz); phospho Thr389 P70S6K (sc-11759, Santa Cruz); total ULK1 antibody (8054 and 4773, Cell Signalling); phospho Ser757 ULK1 antibody (6888, Cell Signalling); Beclin-1 antibody (3738, Cell Signalling); β-actin antibody (A1978, Sigma Aldrich); β-tubulin (T6199, Sigma Aldrich).

Chemical compounds were purchased as follows: LRRK2in1 [24 (link)] from the Division of Signal Transduction Therapy, School of Life Sciences, University of Dundee, U.K.; GSK2578215A and MLi-2 from Tocris [25 (link)]; bafilomycin-A1 (B1793-2UG) and cycloheximide (01810-1G) from Sigma-Aldrich; torin-1 (CAY10997) from Cayman Chemicals. Antibodies used were as follows: LC3 (NB100-2220, Novus Biologicals); total P70S6K (sc-8418, Santa Cruz); phospho Thr389 P70S6K (sc-11759, Santa Cruz); p62 (610833, BD Transduction Labs); total ULK1 antibody (8054 and 4773, Cell Signalling); phospho Ser757 ULK1 antibody (6888 and D7O6UCell Signalling); phospho Ser555 ULK1 antibody (5869, Cell Signalling); total LRRK2 antibody (MJFF2, Abcam); phosphor Ser935 LRRK2 antibody (UDD2, Abcam) and β-actin (A1978, Sigma-Aldrich). AMPK activator (A769662) was kindly provided by Dr MPMS.