A922500

SARS-CoV-2 was originally isolated from nasopharyngeal swabs of confirmed case from Rio de Janeiro/Brazil (GenBank accession no. MT710714). The virus was amplified in Vero E6 cells in high glucose DMEM supplemented with 2% FBS, incubated at 37°C in 5% CO₂ during 2 to 4 days of infection. Virus titers were performed by the tissue culture infectious dose at 50% (TCID₅₀/mL) and the virus stocks kept in -80°C freezers. According to WHO guidelines, all procedures involving virus culture were performed in biosafety level 3 (BSL3) multiuser facility. SARS-CoV-2 infections were performed at MOI of 0.01 in all cells with or without pre-treatment with the pharmacological inhibitor of DGAT-1 (A922500 –Sigma CAS 959122-11-3) for two hours and maintained after the infection. The Plaque-forming Assay was performed for virus titration in VERO E6 cells seeded in 24-well plates. Cell monolayers were infected with different dilutions of the supernatant containing the virus for 1h at 37°C. The cells were overlaid with high glucose DMEM containing 2% FBS and 2.4% carboxymethylcellulose. After 3 days, the cells were fixed with 10% formaldehyde in PBS for 3h. The cell monolayers were stained with 0.04% crystal violet in 20% ethanol for 1h. The viral titer was calculated from the count of plaques formed in the wells corresponding to each dilution and expressed as plaque forming unit per mL (PFU/mL).

GW6471 and Atglistatin were purchased from Cayman Chemical (Ann Arbor, MI, USA). GSK3787 was purchased from Abcam (Cambridge, UK). GW9662 was purchased from FUJIFILM Wako Chemicals (Osaka, Japan). A922500 was purchased from Sigma-Aldrich (St. Louis, MO, USA). Antibodies against carnitine palmitoyltransferase 2 (CPT2), electron transfer flavoprotein subunit α (ETFα), electron transfer flavoprotein dehydrogenase (ETFDH), Acox1, and VLCAD were kindly gifted by T. Osumi (University of Hyogo) [25 (link),26 (link),27 (link)]. ADRP (PRIN2; sc-377429) and adipose triglyceride lipase (ATGL) (sc-365278) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Antibodies against SOX2 (#3579), Nanog (#4903), Oct4 (#2890), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; #5174) were purchased from Cell Signaling Technology (Danvers, MA, USA). Horseradish peroxidase (HRP)-conjugated anti-rabbit IgG and anti-mouse IgG secondary antibodies were purchased from Jackson ImmunoResearch (West Grove, PA, USA).

SCD1 inhibitor (SSI4) was provided by John Copland (Mayo Clinic Florida). DGAT1 inhibitor (A922500) was purchased from Sigma-Aldrich (#A1737). The working concentration for SSI4 and A922500 were 1 µM and 10 µM, respectively. Organoids were treated with inhibitors 48 h prior to being processed for immunofluorescence staining (BODIPY and ER tracker). For in vitro flow cytometry apoptosis assay, the organoids were treated with the inhibitors for 6 days, with the medium changed 3 days after the first treatment and processed as aforementioned.

Lipid
peroxidation was determined after the cells were exposed to silica
NPs for 6 h. Cells were pre-incubated as indicated with 500 μM
Trolox (Sigma) for 1 h at 37 °C prior to addition of the NPs.
Additionally, for some experiments, cells were pre-incubated for 1
h with the DGAT-1 inhibitor A 922500 (Sigma-Aldrich) and/or the DGAT-2
inhibitor PF-06424439 (Sigma-Aldrich) at 5 μM. The cells were
collected and resuspended in PBS containing 2 μM C11-BODIPY
581/591 (Thermo Fisher Scientific). The cells were then incubated
for 30 min in the dark at 37 °C, and the analysis was performed
using the BD LSRFortessa flow cytometer operating with BD FACS-DIVA
software (BD Biosciences). To evaluate the content of lipid droplets,
cells were incubated with BODIPY 493/503 (Thermo Fisher Scientific)
for 15 min at 37 °C. Then, cells were harvested and analyzed
by using the BD LSRFortessa flow cytometer.

All animal protocols were approved by the regional animal study committee of Berlin (Germany) and the Institutional Animal Care and Use Committee of the Institute of Biophysics, Chinese Academy of Sciences. C57BL/6 and BALB/c mice (6–8 weeks of age, female) were purchased from either Janvier labs or the Weitong Lihua Company and were housed under standard conditions with free access to water and autoclaved standard chow. In co‐injection tumor models, 5 × 10⁵ CT26 cells were mixed with indicated amount of purified myeloid cells in Matrigel (BD Bioscience) and then injected subcutaneously. To test the anti‐tumor effect of diacylglycerol acyltransferase (DGAT) inhibitor in vivo, intra‐tumoral injection of DGAT1 inhibitor A922500 (3 mg/kg) and DGAT2 inhibitor PF‐06424439 (10 mg/kg; both Sigma‐Aldrich Chemie GmbH) were administered once daily at day 6 after 5 × 10⁵ CT26 tumor cell inoculation. Tumor growth and body weight were monitored daily. To test the effect of LD formation inhibitors, 2.5 × 10⁵ MCA205 cells were inoculated subcutaneously. Intraperitoneal injection of liposome‐encapsulated inhibitors or liposome vehicle as control started at day 7. Tumor growth was monitored every 2–5 days, and tumor volume was calculated as (length × width × width/2).