The fluorimetry and calorimetry based assays was conducted for characterization of chrysin as HDAC inhibitor.
All cell-based assays were conducted using a human bone-derived cell line (Saos-2), which is recognized to have the capacity to differentiate into mature bone-like cells that produce alkaline phosphatase (ALP) and a bone-like mineralized matrix when cultured under the appropriate mineralizing/differentiating (osteogenic) conditions [41, 42].
These plate-based assays were conducted in triplicate and the clearance zone diameters for skim milk and blood agar plates or the fluorescent halo diameters for the rhodamine B plates were measured and compared by percentage conversion relative to the wildtype PAO1 value and were expressed as an average ± s.e.m (Fig. 7A, 7B and 7C).
The high-throughput cell-based assays were conducted on the HEK293 cell line stably transfected with the HSD11B1 gene using modified literature protocols.
For the thermostability test, the enzyme samples were pre-incubated at different temperatures 35-60°C 35-60°CC increments) at pH 6.0 for a set period of time after which similar DNS-based assays were conducted as described earlier.
The sequences of the primers are listed in Additional file 9. RNAi-based gene-silencing assays were conducted as previously reported: Approximately 69 nl dsRNAs (4 mg/ml) in water were injected into the thorax of cold-anesthetized 4-day-old female mosquitoes using a nano-injector [ 2].
The enzyme assays were conducted based on protocol adapted from the literature, considering several variables [ 12– 12].
Leptin (intra-assay CV, 3.4%, inter-assay CV, 6.4%) (R&D Systems Inc ,Abingdon, UK) and insulin (intra-assay CV, 4.7%, inter-assay CV, 12.5%) (Mercodia AB, Uppsala, Sweden) assays were conducted using serum.
