Filipin complex from streptomyces filipinensis

For confocal microscopy, macrophages were seeded in black poly-d-lysine coated glass 96-well plates (MatTek Corporation, Ashland, MA, USA). To stain lysosomes, macrophages were incubated with 75 nM Lysotracker Red or Deep Red (Thermo Fisher Scientific) at 37°C/5%CO₂ for 1 h before fixation. Cells were fixed for 1 h in 1% EM-grade formaldehyde, followed by quenching with PBS/1.5 mg/ml glycine for 10 min and blocking in 5% human serum for 45 min, all at room temperature. For immunostaining, cells were permeabilized for 10 minutes with 0.1% Triton X-100 before blocking and subsequently stained with primary and secondary antibodies for 30 minutes each in the dark at room temperature. Finally, cells were stained with phalloidin-Alexa488 (Thermo Fisher Scientific) and/or LipidTOX Green (Thermo Fisher Scientific) for 30 min according to the manufacturers’ instructions, and/or 50 μg/ml Filipin complex from Streptomyces filipinensis (Sigma-Aldrich) for 2 h at room temperature in the dark. Lysotracker and filipin pictures were taken using a SP8WLL confocal microscope (Leica, Amsterdam, The Netherlands). Galectin-3 and NDP52 colocalization was visualized using a Dragonfly spinning-disk confocal microscope (Andor Technologies, Belfast, UK) equipped with 405, 488, 561 and 640nm lasers and a Zyla 4.2 sCMOS camera.

Brain and liver tissue from each treatment group at 9-wk-old and end stage were used for immunohistochemical staining. 24–48 h before sectioning of GFP biodistribution samples, tissues were transferred to a 30% sucrose solution where they remained until sinking. Using a cryostat, brain and liver were sectioned parasagittally at a 30 μm thickness and free-floating sections were collected and washed in PBS containing 0.25% Triton X-100 (PBSt). After a 1-h block at room temperature in PBSt/normal goat serum (NGS, S26-M; Sigma-Aldrich), primary antibodies were diluted in PBSt/NGS and sections incubated overnight. Subsequent to washing in PBSt, appropriate secondary antibodies were diluted in PBSt/NGS and incubated with sections for 30 min at room temperature. Refer to Table S2 for antibodies and dilutions. Filipin complex from Streptomyces filipinensis (F9765; 0.025 g/ml; Sigma-Aldrich) was diluted in PBSt to stain tissue for cholesterol accumulation. ProLong Gold mounting medium with or without DAPI (P36930 or P36935; Life Technologies) was used to coverslip slides after sections were mounted. H&E staining was performed by Histoserv, Inc..

ICZ (cis‐4[4‐4‐4[[2‐(2‐4‐dichlorophenyl)‐2‐(1H‐1,2,4,triazol‐1‐methyl)‐1,3‐dioxolan‐4‐yl]‐1‐piperazinyl]phenyl]‐2,4‐dihydro‐2‐(1‐methyl‐propyl)‐3H‐1,2,4‐triazol‐3‐one; MW: 705.64) and ketoconazole were purchased from Sigma‐Aldrich (catalogue numbers #I6657 and #K1003, respectively, St‐Louis, MO, USA), while OSW‐1 ((3β,16β)‐3,17‐dihydroxy‐22‐oxocholest‐5‐en‐16‐yl‐2‐O‐acetyl‐3‐O‐[2‐O‐(4‐methoxybenzoyl)‐β‐D‐xylopyranosyl]‐α‐L‐arabinopyranoside) was from Alfa Chemistry (#ACM145075816, Ronkonkoma, NY, USA). The hydroxy‐ICZ (H‐ICZ), thapsigargin, imipramine and 25‐HC were all purchased from Cayman Chemical (#22576, #10522, #15890 and #11097, respectively, Ann Arbor, MI, USA). Stock solutions were prepared at 10 mM in dimethyl sulfoxide (DMSO) (VWR International, Radnor, PA, USA) for ICZ, H‐ICZ, ketoconazole and thapsigargin; 100 mM for imipramine; 20 mM for 25‐HC; and 1 mM for OSW‐1. Filipin complex from Streptomyces filipinensis was obtained from Sigma‐Aldrich (#F9765), and the stock solution prepared at 25 mg/ml in DMSO. λ‐phosphatase was also purchased from Sigma‐Aldrich (#P9614) at a stock solution of 400,000 units (U) per ml.