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The whole cell assay was done in cultured Human Embryonic Kidney cells (293T), which were treated with inhibitors for 16 h and then incubated with Boc-Ac-Lys-AMC, a membrane permeable HDAC substrate. After 90 min at 37° C, the reaction was quenched with trypsin and TSA by to a final concentration of 1 mg/mL and 1 μM, respectively. The cells were lysed with 1% NP-40. Fluorescence was read at Ex 360 nm, Em 470 nm, using GeminiXS fluorimeter.
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The whole cell assay was done in cultured Human Embryonic Kidney cells (293T), which were treated with inhibitors for 16 h and then incubated with Boc-Ac-Lys-AMC, a membrane permeable HDAC substrate. After 90 min at 37° C, the reaction was quenched with trypsin and TSA by to a final concentration of 1 mg/mL and 1 μM, respectively. The cells were lysed with 1% NP-40. Fluorescence was read at Ex 360 nm, Em 470 nm, using GeminiXS fluorimeter.
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43
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57749198093
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The extent of inhibition of total HDAC activity resulting from treatment of 293T cell with the sulfamide inhibitors was comparable to MGCD0103, which was used as control. It is worth noting that the latter inhibitor resulted in about 80-90% decrease of total HDAC activity in intact cells in comparison to SAHA.
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The extent of inhibition of total HDAC activity resulting from treatment of 293T cell with the sulfamide inhibitors was comparable to MGCD0103, which was used as control. It is worth noting that the latter inhibitor resulted in about 80-90% decrease of total HDAC activity in intact cells in comparison to SAHA.
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57749190647
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note
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The cell-based ELISA assays for tubulin and histone acetylation were conducted according to the following protocol: cells were seeded the day before the treatment in 96-well plates. The following day, the cells were incubated for 3 h with the HDAC inhibitors at different concentrations. The cells were fixed on the plate and the appropriated acetylated histone H3 and tubulin antibody was added. The reaction was developed by Amplex Red. The cell number was normalized by Alamar blue.
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