Kinase experiment:

Phosphatase assays are performed in buffer containing 50 mM Bis-Tris, pH 6.0, 1 mM DTT and 0.01% Triton X-100 at 37°C. For assays conducted with 3-O-methylfluorescein phosphate (OMFP) as substrate, fluorescence is monitored continuously at λex = 485 and λem = 525 nm. For assays conducted with para-nitrophenylphosphate (pNPP) as substrate, the reaction is stopped by addition of 2X reaction volume of 1 M NaOH, and absorbance is measured at 405 nm. IC50 values are determined from plots of LMPTP inhibitor 1 concentration versus percentage of enzyme activity. For inhibitor selectivity assays, each PTP is incubated with either 0.4 mM OMFP or 5 mM pNPP in the presence of 40 μM LMPTP inhibitor 1 or DMSO. Equal units of enzyme activity, comparable to the activity of 10 nM human LMPTP-A, are used. For the inhibitor reversibility assay, 50 nM human LMPTP-A is pre-incubated with 10 μM LMPTP inhibitor 1 or DMSO for 5 min. The enzyme is diluted 100X in phosphatase assay buffer containing 0.4 mM OMFP and fluorescence is measured at the indicated time points[1].

Cell experiment:

Human HepG2 cells are cultured in Eagle’s Minimal Essential Medium (ATCC) containing 10% fetal bovine serum (FBS), 100 U/mL penicillin and 100 μg/mL streptomycin. The absence of Mycoplasma contamination in HepG2 cultures is confirmed using the Lonza MycoAlert Mycoplasma Detection Kit. Cells are treated with 10 μM LMPTP inhibitor 1 in serum-starvation media (0.1% FBS) overnight, following which cells are stimulated with 10 nM bovine insulin for 5 min at 37°C. For detection of IR tyrosine phosphorylation by immunoprecipitation/Western blotting, cells are lysed in radioimmunoprecipitation assay buffer containing 1 mM phenylmethylsulfonyl fluoride, 10 μg/mL aprotinin/leupeptin, 10 mM sodium orthovanadate, 5 mM sodium fluoride, and 2 mM sodium pyrophosphate, and the IR is immunoprecipitated using the anti-IRβ Ab. IR tyrosine phosphorylation of immunoprecipitates is determined by Western blotting with the anti-pIR/pIGFR-Y1162/Y1163 Ab[1].

Animal experiment:

Mice[1]LMPTP inhibitor 1 is administered to male B6 or Acp1fl/fl albumin-Cre+ DIO mice at 0.05% w/w in high-fat diet (HFD) rodent chow. Control groups consist of male B6 or Acp1fl/fl albumin-Cre+ littermate mice administered HFD rodent chow alone. Mice are allowed food and water ad libitum and weighed daily. Randomization is not used in these experiments; rather littermate mice are assigned to treatment or control groups in a manner to maintain similar mean body weights between the 2 groups at the start of the study. Insulin-induced liver IR phosphorylation, IPGTT and fasting insulin levels are assessed after treatment. Diabetic (displaying overnight [13 hr] fasting blood glucose levels ≥140 mg/dL) B6 DIO mice are used in experiments to assess IPGTT and fasting insulin levels[1].

参考文献:

[1]. Stanford SM1, et al. Diabetes reversal by inhibition of the low-molecular-weight tyrosine phosphatase. Nat Chem Biol. 2017 Jun;13(6):624-632.