Pf 429242

Lyophilized lipids, 1-deoxysphinganine (Avanti; cat 860493) and sphinganine (Avanti; cat 860498), were resuspended to stock concentrations at 5 mM in EtOH and subsequently added to retinal organoid culture media at a concentration of 1 μM. For control conditions, equivalent amounts of EtOH were added to the media. ISRIB (Sigma; cat SML0843) was administered at 200 nM. GSK2656157 (Bio Vision; cat 9466) was administered at 500 nM. AA147 was obtained from the Kelly Lab at Scripps Research and resuspended in dimethyl sulfoxide (DMSO); organoids were administered at 10 μM daily. PF429242 (Sigma-Aldrich; cat SML0667) was resuspended in water and administered at 10 μM. CP7 was obtained from the Walter Lab at UCSF, resuspended in DMSO, and administered at 7 μM. 4μ8 C (EMD Millipore; cat 412512) and STF-083010 (Sigma; cat 412510) were resuspended in DMSO and administered at 32 µM. For drug experiments, 1-dSA and drugs were added to organoid culture media at concurrent times. Organoids were cultured in experimental conditions for 4 days unless otherwise stated with a condition-specific media change on day 2. For GSK2656157 and ISRIB experiments, drugs were added on day 0 and day 2. For all other drug experiments, drugs were added daily.

The compound tested in this work, PF-429242, was purchased from Sigma-Aldrich and diluted in deionized water. Amphotericin B, obtained from Cristália, was used as a reference drug in antileishmanial tests against promastigote and amastigote forms, it was also diluted with water and the following concentrations were used in the tests: 0.5, 0.25, 0.12, 0.06, 0.03, and 0.01 μM. Antimycin A was purchased from Sigma-Aldrich and used at 10 μM. Miltefosine was obtained from Cayman Chemical and used at 8 μM.

DHA, AA, OA, PA, betulin, GSK2194069, PF-429242, fluorescein diacetate, and 1,10 phenanthroline were obtained from Sigma Aldrich. C-Laurdan was purchased from TPProbes (South Korea). Amplex Red kit to quantify cholesterol was purchased from Invitrogen. Antibodies used: “actin (1:5000, monoclonal clone AC-15, Abcam), SREBP2 (1:400, polyclonal, Abcam), SREBP1 (1:1000, monoclonal, Abcam), ATF6 mentioned (1:1000, polyclonal, Bethyl Laboratories). Di-4-ANEPPDHQ (Di4), CellEvent™ Caspase-3/7 Green Detection Reagent, Annexin V-Pacific Blue, and Hoechst 33342 purchased from ThermoFisher. Fluorescein sodium salt was purchased from Santa Cruz Biotechnology.

For propionic acid studies, Huh7 cells were cultured in DMEM supplemented with 10% (v/v) LPDS for 24 h and incubated in culture media containing 5 μM statin, the indicated doses of propionic acid sodium salt (Sigma #P5436) or vehicle control (ethanol) for an additional 24 h. For methylmalonic acid studies, Huh7 cells were cultured in Opti-MEM™ with RNAiMAX (ThermoFisher) for 24 h to enhance uptake of methymalonic acid. After this pretreatment, cells were incubated in DMEM containing 10% (v/v) LPDS containing the indicated doses of methylmalonic acid (Sigma #M54058), 5 μM statin or vehicle control (ethanol) for an additional 24 h. Following treatment, gene expression, LDLR activity, total sterol content, and/or ¹⁴C-acetate incorporation into cholesterol were assessed as described. In another set of experiments, cells were pretreated with 10 μM S1P inhibitor (PF-429242; Sigma #SML0667) for 2 h prior to addition of statin or propionate; gene expression was assessed as described below.