Baicalein

Adult male Sprague-Dawley rats were used to induce OA model by DMM surgery (5 groups and in each group n=6; 8-week old; mean body weight=220 g). Briefly, after anesthetization, a medial capsular incision was made to expose the right knee joint. Then the medial meniscotibial ligament was transected, and the medial meniscus could be displaced medially. Finally, the medial capsular incision was sutured, and the skin was closed. A sham operation was performed in another group by only opening the joint cavity. After wound healing, chitosan (Sigma-Aldrich, St. Louis, MO, USA)/siRNA-15-LO-1-CH were formulated as previously described with the final N:P=35.0.^{31 (link)} Then intra-articular injection was performed with 200 μl nanoparticles (0.5 nmol siRNA) twice a week. In addition, 1 mg baicalein (Cayman Chemicals, Ann Arbor, MI, USA) per knee, an inhibitor of 15-LO-1, was intra-articular injected, whereas saline injection was used as control. All animal experiments were performed according to the protocol approved by the Shanghai Jiao Tong University (SJTU) Animal Care and Use Committee.

Oil Red O, 3-isobutyl-1-methylxanthine (IBMX), dexamethasone, and insulin were purchased from Sigma (St. Louis, MO, USA). Baicalein, 2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]-D-glucose (2-NBDG), and Akt Inhibitor X were from Cayman Chemical (Ann Arbor, MI, USA). Anti-Akt and anti-phospho-Akt (p-Akt; Thr308) polyclonal antibodies were obtained from Cell Signaling (Danvers, MA, USA). Anti-C/EBPα (C-18), anti-AMPKα (H-300), and anti-phospho-AMPKα (p-AMPK; Thr172) polyclonal antibodies and normal goat IgG were from Santa Cruz Biotech. (Santa Cruz, CA, USA). Anti-β-actin (AC-15) monoclonal antibody was from Sigma. Horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG antibody was from Santa Cruz Biotech.

Human melanoma cell lines used were: MeWo, A2058, SK-Mel 5, SK-Mel 24, C8161, CHL-1, and A375. Cell identity was confirmed by short tandem repeat analysis (STR) and the DSMZ Online STR Analysis^{52 (link)}, and Mycoplasma testing was routinely performed each month by using the Venor®GeM Classic (Minerva-BiolAbs; Berlin, GE).
Cell lines (BRAF^WT: CHL-1, SK-Mel 24 and MeWo; BRAF^V600E: A375, A2058 and SK Mel-5; BRAF^G464E: C8161) were cultured in DMEM (Sigma-Aldrich; Milan, IT), supplemented with 10% foetal bovine serum (Sigma-Aldrich), 2mM L-glutamine (Sigma-Aldrich), 1% penicillin/streptomycin solution (Sigma-Aldrich) at 37 °C under 5% CO₂. All reagents were purchased from Sigma-Aldrich if not differently indicated. Cells were treated with Thapsigargin 10 μg/ml, Erastin 10 μM, Brusatol 50 nM (Cayman Chemicals; Ann Arbor, MI, US); Medroxyprogesterone 10 μM, Baicalein 20 μM, MG132 10μM, H₂O₂ 500 μM; Deferoxamine 100μM (Cayman Chemicals); Zileuton 10 μM (Cayman Chemicals); Ferrostatin-1 10 μM; Vemurafenib 10 μM; 2-Mercaptoethanol 100μM; RLS3 2μM (Sigma-Aldrich).

Infection experiments were carried out in glass vials containing 6 ml of M9 media with 0.2% glucose supplemented with 100 µM Baicalein (Cayman Chemical) dissolved in DMSO, and 10⁶ cfu/ml P. aeruginosa WT. The same experiment was carried out concurrently with the QS mutant in the presence or absence of 2 µM 3OC12-HSL and 10 µM C4-HSL (Sigma) dissolved in DMSO. Control treatments received the same amount of DMSO. Total amounts of 10⁴ plaque forming units (pfu) (low phage), 10⁷ pfu (mid phage) or 10⁹ pfu (high phage) were added at the same time as the bacteria. Microcosms were incubated at 37 °C while shaking at 180 rpm. Every 24 h microcosms were sampled and transferred to fresh media at a 1:100 dilution. Samples from the infected cultures were taken prior to each transfer and chloroform treated to kill bacteria as described previously [38 (link)]. 5 µl of a serial dilution of chloroform treated sample was spotted onto a lawn of sensitive bacteria (CRISPR-KO strain). Plaques were counted and densities were calculated as pfu/ml. Bacteria were sampled at 3 days post infection to analyse the resistance mechanisms that evolved in response to phage infection.

The human non-small cell lung cancer cells H-460 (large cell carcinoma), A549 (adenocarcinoma) and SKMES1 (squamous carcinoma) were obtained from the American Type Culture Collection (Rockville, MD) and maintained in a humidified atmosphere of 5 % CO₂ in air at 37 °C. They were routinely cultured in RPMI 1640 medium, which was supplemented with 10 % (v/v) foetal bovine serum (Life Technologies Inc.), 2 μM L-glutamine, and 100 μg/ml penicillin-streptomycin. Sub-culturing was carried out when the cells reached 80 % confluency. Baicalein was obtained from Cayman Chemical (Ann Arbor, MI, USA) and made up either in DMSO (in-vitro cell culture studies) or in a solution containing 80 % PBS and 20 % DMSO (in-vivo xenograft studies). Proportionate volumes of DMSO were used for vehicle control groups in all experiments.