Camxc 30

PC3 cells were plated in six-well plates, treated with vehicle or with 20 μM of CUR (or DEX/CUR), and kept in culture. After 24 h, cells were scraped with a p200 tip (time 0 h), washed several times in PBS, and monitored with a phase contrast 10× objective using an Olympus BX41 microscope with CSV1.14 software and a CAMXC-30 for image acquisition. Images were obtained immediately (0 h) and 24 h after scratching, and were representative of the three independent experiments.

Apoptosis was tested by enzymatic labeling of DNA strand breaks using terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL) assay, following the guidelines of the manufacturer (TUNEL assay kit, Promega, Madison, WI, USA), according to Armentano et al. [47 (link)]. Briefly, PC-3 cells were treated with vehicle alone (DMSO) or 10 μM of CUR, 10 μM of QRT, or combined treatment, for 48 h. Then, nuclear staining was achieved using 0.2 mg/mL 4′,6-diamidino-2-phenylindole (DAPI), and apoptotic nuclei were visualized using a fluorescent microscope (Olympus BX4 equipped with CSV1.14 software employing a CAMXC-30 for image acquisition). Unless otherwise indicated, all chemicals were from Sigma/Merck (Darmstadt, Germany).

RAW 264.7 cells were seeded on coverslip in 6-well plates at a density of 1 × 10⁵ cells/well, and cultured overnight in complete medium. Then, they were treated for 1 h with LPS (1 µg/mL) and extract or licoflavanone, using their IC₅₀ values. Next, cells were fixed with ice cold methanol for 20 min at −20 °C, washed three times for 5 min with Tris buffered saline (TBS, Sigma-Aldrich), and incubated for blocking with 5% bovine serum albumin (BSA, Sigma-Aldrich) in TBS for 40 min at 37 °C. Then, cells were incubated for 40 min at 37 °C in anti-NF-kB p65 monoclonal antibody (Santa Cruz, Biotechnology), diluted 1:200. After, they were washed three times for 5 min with TBS to discard the excess of primary antibody, incubated for 40 min at 37 °C in anti-mouse IgG-TRITC (Sigma-Aldrich) diluted 1:300, and subsequently washed three times for 5 min with TBS. Images at 20× magnification were taken on Olympus BX41 microscope with CSV1.14 software, using a CAMXC-30 for image acquisition.

Breast cancer cells were seeded on coverslips in 6-well plates at a density of 1 × 10⁵ cells/well, and cultured overnight in complete medium. Next, cells were treated for 48 h with mevalonate, cholesterol (1 mM or 10 µM, respectively), or EtOH. After treatment had ended, cells were fixed with ice cold methanol for 20 min at −20 °C, washed three times for 5 min with Tris buffered saline (TBS, Sigma-Aldrich), and incubated for blocking with 10% donkey serum (Sigma-Aldrich) in TBS for 40 min at 37 °C. Then, cells were incubated for 40 min at 37 °C in anti-PLIN monoclonal antibody (Abcam), diluted to 1:200, as previously described [34 (link)]. Next, they were washed three times for 5 min with TBS to discard excess primary antibody, incubated for 40 min at 37 °C in anti-rabbit IgG-FITC (Alexa Fluor 488) diluted to 1:500, and subsequently washed three times for 5 min with TBS. DAPI (Sigma-Aldrich, Milan, MI, IT) was also used to stain nuclei. Pictures were taken at 20× magnification using the Olympus BX41 microscope with CSV1.14 software, using a CAMXC-30 for image acquisition.