PA28γ -depletion does not alter the expression level of H2B-GFP or mCherry-H2B, and analysis of re-expression of WT- and Δ C-PA28γ in HeLa H2B-2FPs cells. A. Immunoblot analysis of H2B-GFP and mCherry-H2B expression level in total extracts from parental (WT) and KO-PA28γ HeLa H2B-2FPs cells (left panel). Tubulin was used as a loading control. The relative abundance of HP1 β proteins was quantified using ImageJ software. Graphical representation of the relative abundance of H2B-GFP and mCherry-H2B normalized to tubulin (right panel). The mean ± SD is from four independent experiments. Statistical significance was evaluated based on Student's T-test, ns = not significant. ( p = 0.2027 and 0.4024 for H2B-GFP and mCherry-H2B, respectively) B. Quantification of the H2B-GFP and mCherry-H2B fluorescence intensities in WT and KO-PA28γ HeLa H2B-2FPs cells. The total number of analyzed cells is n = 172 (WT), n = 183 (KO-PA28γ). Statistical significance was evaluated with Student's T-test, ns= not significant. C. FRET analysis in WT, KO-PA28γ HeLa H2B-FPs cells, and WT HeLa H2B-FPs cells treated with Trichostatin A (TSA, 200ng/ml, 24h). The statistical analysis of the mean FRET efficiency percentage is presented as box-and-whisker plots. The thick line represents median, the boxes correspond to the mean FRET values upper and lower of the median, with the whiskers covering the 10-90 percentile range. The total number of analyzed nuclei is n = 154 (WT), n = 132 (KO-PA28γ), and n = 33 (WT + TSA), **** p
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