PS 48

Kinase experiment:

PDK1 activity tests are performed using T308tide as a substrate for PDK1. In brief, PDK1 activity assay is performed at room temperature (22°C) in a 20 μL mix containing 50 mM Tris pH 7.5, 0.05 mg/mL BSA, 0.1% β-mercaptoethanol, 10 mM MgCl2, 100 μM [γ32P]ATP (5-50 cpm/pmol) , 0.003% Brij, 150-500 ng PDK1, and T308tide (from 0.1 to 1 mM). When appropriate, the PDK1 activity assay is performed in a 96 well format and 4 μL aliquots spotted on p81 phosphocellulose papers using ep motion 5070, washed in 0.01% phosphoric acid, dried, and then exposed and analysed using PhosphoImager technology. Activity measurements are performed in duplicates or triplicates with less than 10% difference between replicates. Experiments are repeated at least twice[1].

Cell experiment:

Human renal mesangial cells (HRMCs) are cultured with 1640 media, containing 10% fetal bovin serum at 37 °C in 5% CO2. Cells are cultured with D-glucose at normal (5.5 mM) or high (25 mM) concentrations in serum-free medium. D-Mannitol (25 mM) is used for a control of osmolality. TP is reconstituted in 0.01% DMSO and freshly diluted with culture medium to 10 ug/L before using. To determine the specific role of PDK1 in TP-potentiated anti-proliferation, 5 μM PS48 (MedChem Express, USA) is applied following the treatment of TP. MTT assay is used to detect cell proliferation. HRMCs are seeded at a density of 1x105/mL into 96-well plates. After 12, 24, 48 and 72 h incubation with different compounds, 20 uL MTT (5 mg/mL) is added to each well. Cells are then cultured for an additional 2 h and subsequently lysed using DMSO (150 uL/well). When the formazan crystals completely dissolve, the optical density (OD) is measured at 570 nm. The arithmetic mean OD of six wells for each group is calculated[2].

References:

[1]. Hindie V, et al. Structure and allosteric effects of low-molecular-weight activators on the protein kinase PDK1. Nat Chem Biol. 2009 Oct;5(10):758-64.
[2]. Han F, et al. Triptolide Suppresses Glomerular Mesangial Cell Proliferation in Diabetic Nephropathy Is Associated with Inhibition of PDK1/Akt/mTOR Pathway. Int J Biol Sci. 2017 Sep 21;13(10):1266-1275.