Shrna constructs

shRNA constructs targeting leptin and an shRNA construct targeting a non-human gene serving as a negative control were purchased from SA Biosciences (Frederick, MD, USA). The GFP sequence in the shRNA construct was removed and replaced with dsRed and neomycin resistance, producing a dsRed, neomycin-resistant leptin shRNA construct and a dsRed, neomycin-resistant negative control shRNA construct. The lnASCs (n = 6 donors) and obASCs (n = 6 donors) were transfected with a dsRed, neomycin-resistant leptin shRNA construct or a dsRed, neomycin-resistant negative control shRNA construct using the Neon Transfection System (Invitrogen), using 1400 V for the pulse voltage, 10 ms for the pulse width, and three pulses. Cells were allowed to recover, expanded, underwent antibiotic selection for 2 weeks, and sorted by flow cytometry to verify dsRed expression. Four groups of cells (n = 6 donors/group) were produced: control shRNA lnASCs, leptin shRNA lnASCs, control shRNA obASCs, and leptin shRNA obASCs.

Chemical and standard reagents including filipin and NBD-cholesterol were obtained from Sigma-Aldrich, St. Louis, MO. LysoTracker and fluorescent dextran were obtained from Life Technologies (Grand Island, NY). shRNA constructs were obtained from SA Biosciences (Valencia, CA). Antibodies against tubulin [1:100 for immunofluorescence (IF), T6199, Sigma-Aldrich], EGFP (1:100 for IF, 632380, Clontech) and mCherry (1:100 for IF, NBP1-96752, Novus Biologicals) have been described previously (Whyte et al., 2008 (link); Towns et al., 2009 (link)). Antibodies against StARD9 [1:100 for western blotting (WB), HPA014562] were obtained from Sigma-Aldrich. Site-directed mutagenesis utilized the Phusion kit (New England Biolabs, Ipswich, MA). Antibodies against NPC1 (5 μg/ml for IF, 1 μg/ml for WB, ab108921), LAMP1 (1:10 for IF, ab25630), SREBP1 (1:100 for IF, ab28481) and cathepsin B (1 μg/ml for IF, ab6313) were obtained from Abcam (Boston, MA). filipin (f9765) was obtained from Sigma-Aldrich. NBD-cholesterol (N1148) was obtained from Invitrogen (Waltham, MA). BacMAM-ER (C10590) was obtained from Thermo Fisher Scientific (Waltham, MA). Nocodazole (M1404) and taxol (T7402) were obtained from Sigma-Aldrich. The sequences of oligonucleotides used in this study are provided in Table S3.

The original NPC1 construct was obtained from Dr Ta Yuan Chang (Dartmouth University) and subcloned into pCI-neo (Promega, Madison, WI; E1841), placing the EGFP sequence at the C-terminus using standard methods. A similar approach was used to tag the C-terminus of NPC1 with mCherry. The NPC1 (I1061T) mutant construct was generated by PCR-based mutagenesis. shRNA constructs co-expressing GFP as a transfection reporter were obtained from SA Biosciences. The shRNA #276 (SA Biosciences, KH13606G) anneals to the StARD9 sequence at base pair 8138 and was identified as the most potent in StARD9 depletion. An EST clone encoding the C-terminal ∼1800 aa of StARD9 (KIAA1300) was obtained from Kazusa DNA Research Institute, Chiba, Japan (Nagase et al., 2000 (link)). The remaining portions of StARD9 were cloned by RT-PCR from human cerebellum RNA (Agilent Technologies, Wilmington, DE) and human liver mRNA (Life Technologies). Expression constructs for LAMP1 and cathepsin B were obtained from Michael Overholzer (Memorial Sloan Kettering Cancer Center).