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. 2022 Jan 24;9:801816. doi: 10.3389/fmolb.2022.801816

FIGURE 7.

FIGURE 7

Epigenetic modifications induced by PaDef in Jurkat cells. PaDef induces global acetylation modifications in Histone 3 by inhibiting HDACs. (A) On the left side is a representative image of the western blot. Histones were extracted from cells treated for 24 h with PaDef (47.3 μM) or the corresponding vehicle (DMSO). Proteins were then separated by SDS-PAGE and western transfer assays were performed. Densitometric analysis plots corresponding to western blot analysis are shown on the right side. H3 levels were used as a loading control. Values were normalized to the vehicle (DMSO) for PaDef. ** (p ≤ 0.01) and **** (p ≤ 0.0001) indicate the significant statistical difference based on vehicle Student’s t-test. n = 4. Values represent the mean ± SE of four independent experiments. Vehicle DMSO (0.98%). (B) HDACs activity was measured with Histone Deacetylase (HDAC) Activity Assay Kit (Fluorometric) from Abcam ab156064. After 6 h of treatment with the peptide, nuclear protein extraction was performed and according to the manufacturer’s instructions, basal fluorescence was measured, and, subsequently, measurements were made every 2 minutes for 1 h. Each point shows the mean ± SE of triplicates of three independent experiments. A HeLa nuclear extract (Abcam kit) was used as a positive control for activity, Trichostatin A (TSA) as a control for HDACs activity inhibition, DMSO vehicle (0.98%), and PaDef (47.3 μM). On the right side, the representative image of one of the reaction times (30 min) is shown. Different letters indicate statistically significant differences based on the vehicle (p ≤ 0.05) and were compared using one-way ANOVA and Tukey post hoc test (p ≤ 0.05). (C) PaDef increases Histone 3 methylation marks (dimethylation and trimethylation). On the left side is a representative western blot image. Histones were extracted from cells treated for 24 h with PaDef (47.3 μM). They were subsequently separated by SDS-PAGE and western blot assays were performed. Values were normalized to the vehicle (DMSO) for PaDef. ** (p ≤ 0.01) indicates the significant statistical difference based on vehicle Student’s t-test. n = 4. Values represent the mean ± SE of four independent experiments. Vehicle DMSO (0.98%).