10k mwco

Purified PXR(130-434)-SRC-1 was dialysed overnight against Tris-HCl 20 mM, pH 8.5, NaCl 200 mM, TCEP 1 mM using 10 kDa molecular weight cut-off dialysis cassettes (Slide-A-Lyzer 0.5 ml 10 K MWCO, Thermo Scientific). Protein concentration was determined spectrophotometrically (ɛ_280 nm=26,210 l mol⁻¹ cm⁻¹). Duplicate experiments were performed on Microcal ITC200 (Malvern) operating at 25 °C. Titrations were carried out in Tris-HCl 20 mM, pH 8.5, NaCl 200 mM, TCEP 1 mM supplemented with 0.05% Tween 20 and 5% DMSO (syringe, sample and reference cells). PXR (5 μM) was disposed in 200 μl cell and compounds were delivered from 40 μl syringe. Compound solutions were set to 300 μM when tested individually (Fig. 5b,c), 50 μM each when used simultaneously (Fig. 5f) and 50 μM (EE2, Fig. 5d) or 200 μM (TNC, Fig. 5e) when tested after pre-incubation of PXR with 50 μM TNC or EE2, respectively. Heat exchanges were monitored throughout titrations consisting of 19 injections (one time 0.5 μl followed by 18 times 2 μl) of compound solutions into the cell containing PXR solution. Data analysis and thermodynamic parameter fitting used Microcal Origin software (Malvern).

We obtained cycloheximide, diethylene triamine pentacetate acid (DTPA), N-ethylmaleimide (NEM), iodoacetamide, catalase, palmitic acid, fatty acid-free bovine serum albumin (BSA), protein inhibitors, 2′, 7′-Dichlorofluorescein diacetate (DCFH-DA) fluorescent dyes, Oil Red O, anti-Myc-coupled agarose beads from Sigma; high capacity NeutrAvidin agarose, mini dialysis devices, 10K MWCO, and ECL from ThermoFisher Scientific. We obtained Ni-NTA agarose beads from Qiagen; MG132 from Calbiochem; M-MLV Reverse Transcriptase from Promega; KOD Hot Start DNA Polymerase from Novagen; Taq polymerase, dNTPs, and X-Gal from TaKaRa; Quick change Site-Directed Mutagenesis kit from Stratagene; Cysteine Sulfenic acid probe DCP-Bio1 from Millipore; hydrogen peroxide (H₂O₂) from Sinopharm Chemical Reagent Co.,Ltd.; hematoxylin from Beyotime.

TQ release was investigated in pH 1.0, pH 6.0 and pH 7.4 to simulate the gastrointestinal tract (GIT) pH conditions. Lyophilized TQ-PLGA-PF68 nanoparticles (10 mg) were placed in the dialysis tubing (10 K MWCO, Thermo Scientific, Waltham, MA, USA) and left to submerge in pH 1.0 (0.1 N HCl solution). Later, they were placed in pH 6.0 (PBS solution) and pH 7.4 (PBS solution). Samples were placed in a thermostatic rotary shaker with a speed of 100 rpm at 37 °C. Drug release was measured by sampling 1 mL of the outer media every 30 min intervals. An equal volume of fresh media was then added to replenish total media. The concentration of TQ released from TQ-PLGA-PF68 nanoparticles was measured with a UV spectrophotometer at 257 nm. Results are expressed as a percentage of the cumulative amount of TQ released versus time.
$\mathrm{Cumulative} \mathrm{drug} \mathrm{release} \left(\%\right)=\frac{\mathrm{Mt}}{\mathrm{M}\mathsf{\theta }}\times 100$
Mt is the amount of drug released at time t, and M_θ is the initial drug in nanoparticle formulation. The drug release data were averaged based on three different measurements.