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℃) 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 ℃ 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].

PS48 is an activator of PDK1 with an AC50 of 8 μM.
PS48 activates full length PDK1 (His-PDK1- FL) and PDK1 50-359[Tyr288Gly;Gln292Ala] (His-PDK1 dm) with AC50s (the concentrations required to reach 50% of the maximal activation) of 7.95±0.2 and 10.0±2.0 μM, respectively. PS48 binds to PDK150–359 with a 1:1 stoichiometry and a binding affinity in the micromolar range (Kd=10.3 μM)[1]. PDK1 activator, PS48, has the ability to reverse the cell proliferation inhibition role of triptolide (TP) in vitro. The inhibition role of TP in cell number is significantly reversed by PS48. TP significantly increases the cell proportion in G0-G1 phase and decreases the cell proportion in G2-M and S phase. However, the effect of TP on cell cycle distribution is all reversed by PS48. In addition, suppression of PDK1/Akt/mTOR pathway by TP in high glucose (HG)-treated human renal mesangial cells (HRMCs) is also reversed by PS48, as well as the expression of Ki-67 and proliferating cell nuclear antigen (PCNA)[2].
Reference:
[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.