96 well plate microplate reader

CB1954 kinetic experiments were all carried out in a 96-well microtiter plate (Corning, USA) using a Thermoscientific Varioskan 96-well plate microplate reader [29 (link)]. Product formation at 420 nm was measured over time in order to determine the Michaelis–Menten kinetic parameters of CB1954 against the NfnB-Cys:CPP conjugate. CB1954 (0.1–5 mM), NADH (400 µM) and PB (50 mM, pH 7.2) were combined and incubated at 37 °C for 3 min before purified NfnB-Cys or NfnB-Cys:CPP (1:1 ratio) was added (50 μg/mL; again NfnB-Cys:CPP volume added was adjusted to ensure 50 μg/mL of NfnB-Cys was added). Dimethyl sulfoxide (DMSO) solvent concentration was kept constant at 5% v/v to account for any negative solvent related effect [50 (link)]. Hydroxylamine yield per second was determined by calculating the change in absorbance over 20 s and the molar extinction coefficient, which is the same for both products (ε = 1200 M⁻¹cm⁻¹ at 420 nm) [14 (link),18 (link),39 (link),50 (link),51 (link),52 (link),53 (link)]. Data gathered was transferred to SigmaPlot 12 (SPSS, Systat Software Inc.) where a non-linear regression tool was used to generate a Michaelis–Menten hyperbolic curve and a report containing the kinetic information of the system.

For the resveratrol group, U2-OS cells were inoculated in a 96-well dish at a final density of 5×10³ cells/well and incubated for 24 h. Then cells were treated with resveratrol at varying concentrations (0, 6, 12, 18, 24 μg/ml) for 24, 48 and 72 h. For lentivirus-transduced cells, cell lines stably expressing Cx43 shRNA (shCx43), scrambled shRNA (NTC), and normal U2-OS cells (blank), were grown in 96-well plates for 24, 48, 72 and 96 h (initially planted at 5×10³ cells/well). Thereafter, cell viability was determined by a Cell Counting Kit-8 (Dojindo, Kumamoto, Japan) according to the manufacturer’s instructions, and absorption at 450 nm was determined for each sample using a 96-well plate microplate reader (Thermo Scientific, MA, USA). Cell viability (%) = [OD (treated)-OD (blank)]/[OD (control)-OD (blank)] ×100%.

CB1954 kinetic experiments were all carried out in a 96-well microtiter plate (Corning, USA) using a Thermoscientific Varioskan 96-well plate microplate reader using the method that we have previously described [32 (link),33 (link),34 (link)]. Product formation was measured at 420 nm over time to determine the -Menten kinetic parameters of the CB1954 prodrug with either NfnB-Cys or YfkO-Cys. CB1954 (0.1–10 mM), NADH (400 µM) and PB (50 mM, pH 7.2) were combined and incubated at 37 °C for 3 min before purified NfnB-Cys or YfkO-Cys (10 µg/mL). Hydroxylamine yield per second was determined by calculating the change in absorbance over 20 s and the molar extinction coefficient, which is the same for both products (ε = 1200 M⁻¹ cm⁻¹ at 420 nm) [12 (link),13 (link),15 (link),25 (link),32 (link)]. Data was analysed using SigmaPlot 12 (SPSS, Systat Software Inc., Chicago, USA) where a non-linear regression tool was used to generate a Michaelis-Menten hyperbolic curve and a report containing the important kinetic information of the system under test. Coefficient of Variability (CoV) for the kinetics is 7.06%.