Sodium fluoride naf

Thirty male Swiss albino mice (Mus musculus with 21 days of age, 30 ± 10 g) were randomly divided into three groups (n = 10 per group). The animals were maintained in polypropylene cages (5 per cage), with ad libitum access to food and water, controlled temperature, and humidity and with regular light/dark cycles. The mice exposed to fluoride received deionized water containing 10 or 50 mg/L of fluoride as sodium fluoride (NaF; Sigma Chemical, USA) during 60 days. The nonexposed group (control) received deionized water during the same period. The experimental protocol (register 57-2015) was approved by the Ethics Committee for Animal Experiments of Federal University of Pará, Brazil. At the end of the experiment 60 days, the blood samples were collected from the animals through intracardiac puncture. Then, the blood samples were transferred to tubes for further methodological steps, as described below.

For total cell lysate, cells were lysed using RIPA lysis buffer (50 mM Tris-Hcl pH 7.4, 150 mM Nacl, 1% Triton x-100, 1% Sodium deoxycholate, 0.10% SDS, 1 mM EDTA supplemented with protease and phosphatase inhibtors [1 mM phenylmethylsulfonyl fluoride (PMSF) (Sigma-Aldrich), 2 μg/ml aprotonin (Sigma- Aldrich), 2 μg/ml leupeptin (Sigma-Aldrich), 1 μg/ml pepstatin A (Sigma- Aldrich), 1 mM sodium orthovanadate (Na₃VO₄) (Sigma-Aldrich) and 1 mM sodium fluoride (NaF) (Sigma-Aldrich)] followed by incubation on ice for 30 mins. For mitochondrial and cytosolic fractionation, briefly, 5 × 10⁶ cells were washed in PBS and homogenized in a sucrose isolation buffer (10mM Tris–MOPS, 1mM EGTA/Tris, 200mM sucrose, pH 7.4), with a Dounce homogeniser. The homogenised mix was then centrifuged at 600g for 10min at 4°C. Supernatant (containing soluble cytoplasm and heavier membrane organelle) was collected and centrifuged at 7,000g for 10min at 4°C. The supernatant, which constitutes the cytoplasm and other heavy organelles, was collected as a separate fraction for further quantification and analysis. The pellet was separately washed and centrifuged at 7,000g for 10min at 4°C to obtain a pure pellet with mitochondria. It was then lysed using RIPA lysis buffer as above.

Palmatine (Pal, CAS number: 3486-67-7, the structure shown in Fig 1) was purchased from Sichuan Pure Chemical Industries (Sichuan, China). Metronidazole (Met, CAS number: 443-48-1,) was purchased from TOKU-E Company (Tokyo, Japan). Acetohydroxamic acid (AHA, CAS number: 546-88-3, purity: 98%), urea (Molecular Biology Reagent), jack bean urease (JBU, type III with specific activity 40.3 U/mg solid), HEPES (Amresco > 99%), L-cysteine (L-cys), glutathione (GSH), dithiothreithol (DTT), boric acid (BA) and sodium fluoride (NaF) were all purchased from Sigma-Aldrich (St Louis, MO, USA). Bradford Protein Assay Kit was purchased from Beyotime Institute of Biotechnology (Shanghai, China). HEPES buffer (20 mM, pH 7.5) was prepared by adjusting the pH with NaOH. Other chemicals and solvents were of analytical grade or HPLC grade and obtained from Guangzhou Chemical Reagent Factory (Guangzhou, China).

Andrographolide sodium bisulfite (C₂₀H₂₉O₇S · Na, CAS number 71202-97-6), urea (molecular biology reagent), D,L-dithiothreitol (DTT) , L-cysteine (L-cys), boric acid and sodium fluoride (NaF) were purchased from Sigma Aldrich. JBU (from jack bean, Canavalia ensiformis, type III, nominal activity 40.3 units/mg, solid) was also from Sigma Aldrich, of which one unit of urease activity is defined as the amount of enzyme needed to liberate 1.0 μmol of NH₃ from urea per min at pH 7.0 at 25 °C. Brucella broth was purchased from Becton–Dickinson. (Cockeysville, MD). Other chemicals were obtained from Guangzhou Chemical Reagent Factory (China). All reagents were of analytical grade. Phosphate buffer (PBS, 20 mM, pH 7.0) was prepared by adjusting pH of phosphoric acid with NaOH. 2 mM EDTA was added to all enzyme-containing solutions.

KN62 (Tocris, #1277), PD98059 (Tocris, #1213), KT5823 (Tocris, #1289), Chelerythrine chloride (Tocris, #1330), H-89 dihydrochloride (Calbiochem, #371963), Rapamycin (Tocris, # 1292); Actinomycin-D (Tocris, #1229), Anisomycin (Sigma, #A9789) Sodium Fluoride (NaF; Sigma #S7920), Phenylmethylsulfonyl Fluoride (PMSF; Sigma, #P-7626) Okadaic Acid (Tocris, #1136).

EOC cells were plated at 2 × 10⁵ cells/well in a 6-well plate. Cells were treated with CDDP, CQ, or CQ-CDDP combination at the indicated concentrations for 24 h or 48 h. Cells were lysed in RIPA buffer containing 0.1 M sodium fluoride (NaF, Sigma), 0.1 M β-glycerophosphate (Sigma), protease inhibitor cocktail (Sigma), and 1 mmol/L phenylmethylsulfonyl fluoride (PMSF, Sigma). Western blot analysis was performed as previously described^{33 (link)}. Primary antibodies against ATM (sc-23921), Cdc2 (sc-8395), Keap1 (sc-365626), and β-actin (sc-47778) were obtained from Santa Cruz Biotechnology (TX. USA); antibodies against p-ATR (#2853), ATR (#13934), p-Cdc2 (Tyr-15,#4539), p21^WAF1/CIP1 (#2947), p-Akt (#9271), and Akt (#9272) were from Cell Signal Technology (MA, USA); antibodies against Cyclin B1 (ab32053), p-ATM (S1981, ab81292), and p62/SQSTM1 (ab56416) were from Abcam; and antibodies against γH2AX (NB100-384), LC3B (NB100-2220), and ATG5 (NB110-53818) were purchased from Novus (CO, USA). Antibody for Nrf2 was purchased from Thermo Fisher (PA5-27882).