M1000 pro microplate reader

This method followed the protocol established by Rothan et al. [51 (link)] with modification. Briefly, a total volume of 100 μL of the reaction contains 3.5 μM purified NS2B-NS3 protease in the standard buffer solution at pH 8.5 (200 mM Tris-HCl) with different concentrations of synthesized imidazole phenazine derivatives in the range of 5.5–700.0 μM were preincubated at 37 °C for 10 min. Subsequently, 150 μM substrate was added and the assay mixtures were incubated at the same temperature for another 1 h. The assay condition was validated by performing both a blank control (100 μL of pH 8.5, 200 mM Tris-HCl buffer only) and a negative control (100 μL 3.5 μM DENV NS2B/NS3 protease and 150 μM substrate Boc-Gly-Arg-Arg-MCA in standard buffer) were carried out simultaneously. The experiments were performed in triplicate (n = 3). As above, the assay was performed using a black 96-well flat-bottom TC multiplate plate format on a Tecan M1000 PRO microplate reader at excitation and emission wavelengths of 365 and 410 nm, respectively. The fluorescence signal was then used to calculate the percentage of inhibition activity of the peptide inhibitors as Equation (1). $\%ofenzymeactivity=\frac{sample-buffer}{negativecontrol-buffer}\times 100$ $\%ofinhibition=100-\%ofenzymeactivity$

U2OS and HCT116 were transfected with 5 nM of respective siRNAs using RNAiMAX. The next day, cells were seeded in 96-well plates (9000 cells per well). After 24 h of transfection, cells were treated with Doxorubicin, 5-FU, Nutlin-3a, or DMSO control for 24 h. Subsequently, the cells recovered for 6 days in fresh drug-free media. WST-1 reagent (Sigma Aldrich) was added for 2 h following the manufacturer protocol before absorbance was measured at 440 nm on a M1000pro microplate reader (Tecan, Männedorf, Switzerland).