Curcumin

Quercetin (≥95%; Santa Cruz Biotechnology, Dallas, TX, USA) and curcumin (≥95%; Santa Cruz Biotechnology, Dallas, TX, USA) were added to NOW diet at various concentrations (Section 2.5) using alpha cellulose (Santa Cruz Biotechnology, Dallas, TX, USA) as a carrier. For 1.0 mmol kg⁻¹ Quercetin (MW = 302.2), 252 mg of Quercetin was weighed into 25 ml volumetric flask and dissolved in acetone (Fisher Scientific, Waltham, MA, USA). Three ml of the solution was added to 2 g of alpha cellulose and stirred to evenly distribute the Quercetin until the acetone was evaporated. The Quercetin coated alpha cellulose was then incorporated in 100 g NOW diet by stir mixing. Other concentrations of Quercetin incorporated diet were made from the dilutions of the 25 ml acetone solution following the same procedures. To generate 1.0 mmol kg⁻¹ curcumin (MW = 368.4), 307 mg of curcumin was weighed into a 25 ml volumetric flask and dissolved in acetone. The same procedures were then followed to incorporate curcumin into 100 g NOW diet.

Cells were pre-treated with Tip60 inhibitors, MG149 (Axon MedChem) or NU9056 (Tocris Bioscience), or general HAT inhibitors, curcumin (Santa Cruz Biotech) or garcinol (Santa Cruz Biotech), for 6 h. KSHV reactivation was induced by treating HEK293T.Bac36 cells with 20 ng/mL 12-O-Tetradecanoylphorbol-13-acetate (TPA) (Sigma-Aldrich) and 1.5 mM sodium butyrate (SB) (Sigma-Aldrich). For MC116.219, BCBL-1, and BC-3 cells, 0.3 mM of SB was used instead.

To evaluate the response to oxidative stress, stem/precursor thyroid cells and mature thyrocytes were exposed for the indicated times and concentrations to the free-radical-generating agent menadione (Vitamin K3, Sigma-Aldrich), a synthetic analog of 1,4-naphthoquinone with redox cycling activity used in many studies to induce oxidative stress [33 (link),34 (link)]. Stock solutions were freshly prepared in DMSO at 5.8 mM menadione, and working solutions were prepared in RPMI medium. Biological changes due to menadione were also evaluated in the presence of 5 µM of curcumin (Santa Cruz Biotechnology, Dallas, TX, USA), a phytochemical with protective activity against oxidative stress [36 (link),37 (link)].

Details of the animal study, including curcumin dosage details, and the animal diet used in this project were reported previously by our group [3 (link)]. Briefly, five-week-old male B6 mice (Jackson Laboratory, Bar Harbor, ME, USA) were single-housed in specific-pathogen-free (SPF) cages at 23 °C. The animal study protocol was approved by the Texas Tech University Institutional Animal Care and Use Committee (TTU protocol numbers16011-04 and 19034-04). The mice were separated into two groups: HF and HFC (n = 8–10 per group). The food composition for both groups contained 45% kcal fat, 35% carbohydrates, and 20% protein. However, only the HFC group’s food was prepared with 0.4% (w/w) curcumin (98.24% pure) (Santa Cruz Biotechnology Inc., Dallas, TX, USA). After 14 weeks of feeding, the mice were fasted for 5 h before euthanasia by CO₂ asphyxiation. Their blood was collected using cardiac puncture followed by serum collection using polymer gel containing BD microtainers (Becton, Dickinson & Co, Franklin Lakes, NJ, USA). The collected tissue samples were flash-frozen in liquid nitrogen. The serum and snap-frozen samples were stored at −80 °C until analysis.

The ATM kinase inhibitor (ATMin) 2-morpholin-4-yl-6-thianthren-1-yl-pyran-4-one (KU55933) and Chk2 kinase inhibitor (Chk2in) 2-(4-(4-chlorophenoxy)phenyl)-1H-benzimidazole-5-carboxamide, were purchased from EMD4 Biosciences (Gibbstown, NJ, USA). Caffeine was purchased from Sigma Aldrich. Curcumin was purchased from Santa Cruz Biotechnology (sc-200509). All inhibitors were prepared in 10 mM stock solution dissolved in DMSO.

Curcumin (1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione) was purchased from Santa Cruz (Santa Cruz Biotechnology, Santa Cruz, CA, USA). MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-zolium bromide, a yellow tetrazole], DMSO (dimethyl sulfoxide), RNase, and propidium iodide were obtained from Sigma (Sigma, St. Louis, MO, USA). Dulbecco’s Modified Eagle’s Medium (DMEM), fetal bovine serum (FBS), and trypsin/EDTA were purchased from Gibco (Pittsburgh, PA, USA). RNX-Plus Solution and the First Strand cDNA Synthesis Kit were purchased from Sinaclon (Sinaclon, Tehran, Iran). Finally, 5-fluorouracil (5-FU) was provided by EBEWE PHARMA (EBEWE Pharma, Unterach, Austria).

SB203580, which inhibits p38 MAPK activity, was obtained from Cell Signaling. The NFκB inhibitor, curcumin, was obtained from Santa Cruz Biotechnology. Interleukin-1 beta neutralizing antibodies (specific for mature mouse or mature human IL-1 beta) and the rabbit polyclonal isotype control antibodies were obtained from Abcam.