Synergy h1 hybrid multi

HeLa cells grown in 12-well plates were infected with RLuc-CVB3 at an MOI of 1. Immediately after infection, DDD85646 (5 μM) or GuHCl (2 mM) was added. Addition of DMSO (0.1% final concentration) served as solvent control for the NMT inhibitor. At 7 h p.i., cells were lysed for determination of Renilla luciferase activity using the Renilla Luciferase Assay System (Promega) according to the manufacturer´s instructions. Luminescence was detected with the Synergy H1 hybrid multi-mode microplate reader (BioTek). The increase of relative light units (RLU) in the absence of GuHCl was taken as measure of RNA replication.

Cell proliferation was assessed by the Cell Counting Kit-8 (CCK-8; Vazyme). Briefly, 2×10³ cells/well were plated in 96-well plates and 10 μL CCK8 solution was added into each well 2 h before detection at the indicated time points. The absorbance at 450 nm (A450) was examined by Synergy H1 hybrid multi-mode reader (BioTek, USA).

GA concentration was determined according to manufacturers' instructions using a competitive ELISA kit (Human glycated albumin ELISA Kit, CSB-E09599h, Cusabio, Wuhan, Hubei Province, China) [14 (link)]. Samples were diluted to 1:250 with the sample diluent buffer provided with the kit to achieve sample absorbance within the range of a standard curve. The absorbance was measured at 450 nm using a Synergy H1 hybrid multi-mode microplate reader (Biotek, Winooski, VT, USA). The amount of GA was determined by comparing with the known standard provided with the kit and expressed as nM/ml of GA present in human serum samples.

The cytotoxicity effect of GPE was studied by MTT assay. The CHMp‐13a and CHMp‐5b cells (7 × 10³ cells/well) were seeded into 96‐well plates and allowed to grow for 24 h. They were then treated with GPE at various concentrations (40, 80, and 160 μg/ml) for 0, 17, 21, and 28 h, whereas the negative control group was treated with the culture medium containing DMSO (0.1%). After incubation, 10 μl of MTT solution was added to each well and the plate was further incubated for 4 h. Then, 100 μl of solubilization solution was added to each well to solubilize water insoluble purple formazan crystals, followed by overnight incubation at 37°C in 5% CO₂. The absorbance at 570 nm was measured using a Synergy H1 hybrid multi‐mode microplate reader (BioTek, Winooski, VT, USA). The experiment was performed in three replicates. The results of absorbance values of treated cells were expressed as percentage of cell viability relatively to untreated control cells (considered as 100% viability).