Pf 06424439

The combination of diacylglycerol acyltransferase DGAT inhibitors (iDGAT) (DGAT 1 inhibitor: A922500; DGAT2 inhibitor: PF-06424439, both Sigma-Aldrich Chemie GmbH) served to block the lipid droplets formation. MSC-2 cells were pre-incubated with 100 nM iDGAT for 1 h, and then cultured with the indicated FFA in the presence of 100 nM iDGAT for additional 24 h.

Cells were seeded at 2000 cells per well on a 96-well plate and were grown overnight at 37 °C. The following day, cells were treated with the following drugs at indicated concentrations for 72 h for IC₅₀ assays: erastin (Cayman Chemicals), RSL3 (Apex Bio), TBH(Sigma), nutlin-3a (Calbiochem), cisplatin (Acros Organics), Etoposide (Sigma), Camptothecin (Cayman Chemicals), and doxorubicin (Cell Signaling). Because fetal bovine serum and culture media can contain significant levels of glutathione, pyruvate, and other agents that can influence sensitivity to ferroptosis (10 (link)), all ferroptosis sensitivity assays were performed in low serum/low glucose (2% fetal bovine serum, DMEM low glucose (Corning Cellgro, 1 g/l glucose)). For rescue assays, cells were pretreated with ferrostatin (Cayman Chemicals) or liproxstatin (Sigma) for 30 min prior to treatment with RSL3 for 24 h. For lipid droplet assays, cells were pretreated overnight with oleic acid (Sigma, O3008), the DGAT2 inhibitor PF-06424439 (Sigma, PZ0233), or vehicle (fatty-acid–free BSA), and viability was assayed using Trypan Blue (Corning, 25-900-CI) after 72 h. At assay endpoint, cells were treated with Alamar blue (Life Technologies Dal1025) for 3 h at 37 °C and absorbance was read using a SynergyHT plate reader (BioTek). GraphPad Prism software was used to perform the data analysis.

PF-06424439 was obtained from Sigma-Aldrich (Saint Louis, MO, USA, #PZ0233). The powder was dissolved in sterile distilled water to make a stock concentration of 18.65 mM and stored at −20 °C in small aliquots. The effect of PF-06424439 on MCF7 cells was tested at 10 µM by dissolving the appropriate volume of the stock solution in complete medium. PF-06424439 solutions were freshly prepared for each experiment.

HUVEC were a kind gift of Špela Zemljič Jokhadar (Institute of Biophysics, Medical Faculty, University of Ljubljana, Ljubljana, Slovenia). Dulbecco’s Modified Eagle Medium (DMEM) high glucose, fetal bovine serum (FBS), Hanks’ balanced salt solution (HBSS), Dulbecco’s phosphate buffered saline (DPBS), L-glutamine, fatty acid free bovine serum albumin (FAF-BSA), NAC, α-tocopherol, resazurin sodium salt, NR, 7-aminoactinomycin D (7-AAD), Hoechst 33,342, T863, PF-06424439, cacodylic acid, sodium hydroxide, ethanol, acetic acid, sucrose, osmium tetroxide, formaldehyde, and glutaraldehyde were obtained from Sigma Aldrich (St. Louis, MO, USA), TrypLE Select and CyQuant Direct Cell Proliferation Assay Kit from Life Technologies (Carlsbad, CA, USA). OA was from Cayman Chemical (Ann Arbor, MI, USA), BODIPY 493/503 and CM-H₂DCFDA from Thermo Fisher Scientific (Waltham, MS, USA).

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.