Sodium Palmitate

The cellular FAO activity was determined by quantifying the conversion of ³H palmitic acid to ³H₂O over the labeling time. Cells were seeded in 12-well plates and when appropriate, treated with FAO inhibitors or stimulators. For FAO assays in complete medium with 10% FBS, [9,10-³H(N)]-palmitic acid [0.5 µCi (~9.3 pmol)] in 15 µl carrier solution (sodium palmitate 1 mM/BSA 0.17 mM/NaCl 150 mM)^{76 (link)} was added to each well (final concentration of added palmitate was ~15 µM, in addition to free fatty acids and other lipids present in FBS). For FAO assays in Krebs buffer or culture medium without FBS, the volume of carrier solution was increased to 50 µl, making a final concentration of palmitate at ~50 µM. After labeling, culture medium (0.5 ml out of 1 ml) was collected into a 15-ml Falcon polypropylene conical tube (#352096, BD Biosciences, San Jose, CA), and mixed with 100 µl of 1.2 N HCl to terminate all biological reactions. The 15-ml Falcon tube has a longer screw top to ensure tight closure than similar products of other brands. A 0.5-ml microcentrifuge tube containing 0.25 ml of sterile distilled water was uncapped and inserted into the 15-ml tube with forceps. Precautions were taken to prevent direct contact between the medium and water phases. The 15-ml tubes were tightly capped to allow diffusion between two liquid phases at room temperature for 3 days unless otherwise indicated.
To control for background from diffusion of ³H-palmitic acid, each experiment included a cell-free blank control with the same medium and water settings. When the diffusion of ³H₂O reached equilibrium between the water and medium, 0.25 ml water or 0.25 ml medium (out of 0.6 ml) were mixed with 3 ml of scintillation cocktail and the radioactivities present in the 0.25 ml water (a) and 0.6 ml medium (b) were determined by scintillation counting. The ³H₂O-based radioactivity in the whole sample would be $\frac{(250ul+600ul)}{250ul}$  × a = 3.4a, where the total radioactivity of the sample would be a + b. The conversion of ³H-palmitic acid to ³H₂O was calculated from the formula $\frac{3.4\mathit{a}}{\mathit{a}+\mathit{b}}$ . By subtracting the mean of the control group, the FAO rate for each sample would be ${\left(\frac{3.4\mathit{a}}{\mathit{a}+\mathit{b}}\right)}^{sample}-{\left(\frac{3.4\mathit{a}}{\mathit{a}+\mathit{b}}\right)}^{blankmean}$ . The values were normalized to cell numbers and labeling times, and presented as % conversion/2 × 10⁵ cells/5 hours in most cases or % conversion/2 × 10⁵ cells/2 hours for the metabolically active hepatocytes and cardiomyocytes.

Immortalized mouse podocyte cell line was a kind gift from Professor Peter Mundel (Harvard Medical School, Charlestown, MA, USA). These cells were grown on collagen I (Catalog # A10483-01, Life Technologies Corporation, Grand Island, NY, USA) coated dishes with low glucose (5.5 mM) Dulbecco's modified Eagles medium supplemented with 5% fetal bovine serum, 100 U/ml penicillin and 100 μg/ml streptomycin at two different temperatures.^{49 (link), 50 (link)} At 33 °C, cells are allowed to proliferate (permissive condition) in the presence of 20 U/ml mouse recombinant IFN-γ (R & D Systems, Minneapolis, MN, USA). For the induction of differentiation (non-permissive condition), podocytes were thermo-shifted to 37 °C in the absence of IFN-γ for 14 days. Expression of synaptopodin, a specific marker for podocyte differentiation, was gradually increased during culture in non-permissive conditions (37 °C), whereas it was not detected in podocytes maintained under permissive conditions (33 °C with interferon-γ).
Palmitate and oleate were prepared by conjugation with fatty-acid-free BSA. First, sodium palmitate or sodium oleate (100 mM) was dissolved in autoclaved distilled water (DW) at 70 °C for 30 min. BSA (10%) was also dissolved in autoclaved DW at 55 °C for 30 min and filtered. Dissolved palmitate or oleate solution was added dropwise into the BSA solution and stored as a stock (10 mM) at −80 °C until use. KRB solution contained (in mM) 135 NaCl, 3.6 KCl, 2 NaHCO₃, 0.5 NaH₂PO₄, 0.5 MgSO₄, 1.5 CaCl₂, 10 HEPES, 5.5 glucose (pH 7.4, 318 mOsm/kg H₂O). 2′,7′-Dichlorofluorescein diacetate DCF-DA, mitoSox, JC-1 and TMRM were purchased from Molecular Probes (Invitrogen, Grand Island, NY, USA). SSO was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and mitoTEMPO was purchased from Enzo Life Sciences (Enzo Life Sciences, Inc., Farmingdale, NY, USA). Sodium palmitate, sodium oleate, fatty-acid-free BSA, MTT, 4′,6′-diamidino-2-phenylindole (DAPI), edelfosine, DKI R59022, GF109203X and other drugs were purchased from Sigma (St. Louis, MO, USA).

Withaferin A was purchased from Xenon Biosciences (India), and the chow diet was procured from Adita Biosys Pvt Ltd. The high-fat diet was procured from VRK Nutritional Solutions, India. Glucose and fructose were procured from Sisco Research Laboratories Pvt. Ltd. Commonly used liver function test enzymes, like aspartate transaminases or aspartate aminotransferase (AST), alanine transaminase or alanine aminotransferase (ALT), and alkaline phosphatase (ALP), and lipid molecules like total cholesterol, triglycerides (TG), and high-density lipoproteins (HDL) kits were purchased from Agape Diagnostics Ltd. TRIzol reagent, sodium palmitate, oleate and Oil Red O stain solution were purchased from Sigma Aldrich, St. Louis, Missouri, United States. cDNA synthesis and SYBR green kits were purchased from Thermo Fisher Scientific. A TGF-β1 ELISA kit was purchased from Krishgen Biosystems. HepG2 and Huh7 cells were purchased from NCCS Pune, India. The cell culture media Minimum Essential Medium Eagle (MEM), Ham DMEM/F-12, 1:1 mixture, bovine serum albumin, and hematoxylin were purchased from HiMedia, India. Fetal bovine serum (FBS) and antibiotics like penicillin/streptomycin were purchased from Gibco. 25-Hydroxycholesterol was a gift provided by Dr. Perumal Madan Kumar, CSIR-CFTRI, Mysuru. Taurochenodeoxycholic acid and deoxycholic acid were a gift from Dr. Ramprasad Talahalli, CSIR-CFTRI, Mysuru.

Stock solutions of sodium palmitate (SP) and oleate (OA) (Sigma-Aldrich, United States) were prepared, as previously described (Römer et al., 2021 (link); Cao et al., 2012 (link)). Briefly, 100 μM of SP and OA were incubated for 30 min at 50 °C. Later, fatty acids were mixed with BSA in a culture medium (the fatty acid to BSA molar ratio was 4:1). To induce steatosis, HepG2 and Huh7 cells were exposed to SP and OA conjugated with fatty acid-free BSA. After incubation for 24 h, the cells were treated for 24 h with various concentrations of withaferin A (1, 2.5, and 5 μM). Cells used as controls were treated with fatty acid-free media containing ethanol as a vehicle.

Cell lines were obtained from the Cell Bank of Chinese Academy of Sciences (Shanghai, China). Huh7 and HepG2 cell lines were grown in RPMI1640 or high-glucose DMEM medium (Gibco, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco, USA) and 100 U/ml penicillin and 0.1 mg/ml streptomycin (MI00614, Mishushengwu, Xi’an, China). AML12 cell line was grown in DMEM: F12 medium (11330, Invitrogen, USA) supplemented with 10% FBS, 1% ITS liquid media supplement (I3146, Sigma, USA), and 40 ng/ml dexamethasone. All cell lines were kept in a humidified incubator at 37 °C and 5% CO₂. Cells were used from third to tenth passage in each experiment. Primary hepatocytes were isolated from 8-week-old male C57BL/6 mice and were cultured in HM medium (ScienCell, USA) and seeded in six-well plates at 1 × 10⁶/well. To induce cellular lipotoxicity, 0.3 mM sodium palmitate (SYSJ-KJ, Kunchuang Biotechnology, Xi’an, China) was added into medium and vehicle was added as a control. After 36 h, tamoxifen dissolved in DMSO was added at a dose of 10, 20, 40 μM and kept for 36 h and DMSO was added as a control. To induce JNK activation in vitro, anisomycin (10 μM) was added 2 h before tamoxifen treatment. To inhibit JNK phosphorylation in vitro, 30 μM Tanzisertib (cc-930) (S8490, Selleck, China) was added 2 h before tamoxifen treatment. For cellular Oil Red O (ORO) staining, cells were cultured in six-well plates and after sodium palmitate and tamoxifen treatment, culture medium was removed, and cells were fixed with 4% paraformaldehyde for 30 min and washed with PBS three times. Then the cells were treated with 60% isopropanol for 5 min. Remove isopropanol, stain cells with ORO working solution (Servicebio Technology, Wuhan, China) for 10 min, and washed cells with PBS. Then the cells were stained with hematoxylin for 3–5 min and washed with PBS at least three times. Then we observed and took photos using an inverted phase contrast microscope (Olympus, X71, Japan). For cellular TC and TG tests, we purchased commercial kits from Pulilai Gene Technology Co., Ltd (Beijing, China) and followed the manufacturer’s instructions.