Use of Agrimol B as an Inducer of TFEB Nuclear Translocation
A technology of Agrimony phenol and inducer, applied in the application field of inducer, can solve problems such as functional deficiency and abnormal cholesterol metabolism
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Embodiment 1
[0056] Example 1: Formulating Compounds
[0057] (1) Preparation of Agrimol B: Weigh Agrimol B powder and dissolve it in DMSO to prepare a 10 mmol / L stock solution, and store at -20°C. In vitro cell experiments were used after dilution with DMEM complete medium.
[0058] (2) Preparation of rapamycin (rapamycin): Weigh rapamycin powder and dissolve it in DMSO to prepare a 10 mmol / L stock solution, and store at -20°C. In vitro cell experiments were diluted to 1 μM with DMEM complete medium for use.
[0059] (3) Preparation of Torin 1: Weigh Torin 1 powder to prepare a 1 mmol / L stock solution and store at -20°C. Dilute to 250nM with DMEM complete medium for in vitro experiments.
[0060] (4) Preparation of SAHA solution: weigh SAHA powder to prepare a 100 mmol / L storage solution, and store at -20°C. In vitro experiments were used after dilution with DMEM complete medium.
Embodiment 2
[0061] Example 2: Cell Culture
[0062] U2OS-TFEB-EGFP cells were cultured in complete DMEM medium, and when the growth state was good, they were seeded on 96-well black-walled transparent bottom plates at the number of 11,000 cells / well and cultured overnight. On the next day, the compound to be tested was added and incubated for 4 hours to detect TFEB nuclear translocation.
[0063] The U2OS-mCherry-EGFP-LC3 stable expression cell line was cultured in a medium containing 2 μg / mL puromycin until the growth state was good, and 6500 cells / well were seeded on a 96-well black-walled transparent bottom plate. DMEM complete medium for overnight culture. On the next day, four hours after adding the compound to be tested, the expression level of LC3 was detected.
[0064] GM03123 cells were cultured in EMEM medium containing 10% fetal bovine serum. In the logarithmic growth phase, cells were seeded on 96-well black-walled transparent bottom plates at 2400 cells / well, and cultured ...
Embodiment 3
[0066] Example 3: Study on the nuclear translocation of TFEB induced by Agrimol B
[0067] Dilute Agrimol B to concentrations of 200 μM, 100 μM, 50 μM, 25 μM, 12.5 μM, 6.25 μM, 3.125 μM, and 1.5625 μM, respectively pipette 20 μL into U2OS-TFEB-EGFP cells containing 180 μL of medium, so that the final concentration 20 μM to 156.25 nM, respectively. In parallel experiments, 1 μM rapamycin and 250 nM Torin 1 were used as positive controls. Cells in the non-drug treatment group were used as negative controls. At the same time, the medium without glucose, without glucose and glutamine was used as the control of nutrient starvation. After drug incubation for 4 h, the cells were fixed with 4% paraformaldehyde. Cell nuclei were labeled with 10 μg / mL Hoechst 33342, and 100 μL of fluorescent dye was taken and incubated with the cells for 15 minutes. Rinse 3 times with PBS, collect TFEB and nuclei fluorescence pictures with MD ImageXpress. Such as figure 2 As shown, agriculol B ca...
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