Fig 2: Moscatilin reduced the protein level of HDAC3. (A) Protein levels of HDAC1, HDAC2 and HDAC3 were determined by western blot after the treatment of moscatilin for 48 h. (B-D) Statistic analysis of protein levels of HDAC1, HDAC2, and HDAC3 performed in panel (A). n = 3. *** indicated P < 0.001 compared to the control group.

Fig 3: Cellular localization of HDAC2 in patient derived lymphoblastoid cell line (LCL). (A) 60x confocal images showing an example of HDAC2 immunocytochemistry on HD LCL and patient LCL (#249); HDAC2 is marked with green signal and nuclei with blue (DAPI); Insets show cell magnification of degree 4 (in HD box) and degree 0 (in #249 box) of HDAC2 nuclear localization. (B) Schematic representation of degrees set for evaluation of HDAC2 presence in nucleus, from absent (Degree 0) to most abundant (Degree 4). (C) Frequency of HDAC2 positive cells (% HDAC2 + cells, on Y-axis) into the five degrees of nuclear localization (in shapes from green for Degree 4 to blue for Degree 0) observed in patient (#249) and HD LCLs (on X-axis); multiple t-test were used as statistical method (* p < 0.05; ** p < 0.01; *** p < 0.001). (D) Western blot of HDAC2 protein (55 kDa) in control (HD) and patient (#249) LCLs lysates fractionation (total, C = cytoplasmic fraction, N = nuclear fraction) compared to cytoplasmatic marker HSP90 (90 kDa) and nuclear marker histone H3 (17 kDa). (E) Quantification of HDAC2 relative enrichment in HD (grey bar) and #249 (red bar) LCLs; all values are expressed as means ± SEM and data analysis was performed using Student’s t-test as statistical method, with p value < 0.05

Fig 4: Whole-transcriptome RNA-seq analysis performed on LCLs from patient #249, RSTS and controls. (A) Heatmap of RNA-seq expression data showing top 50 DEGs in two controls (HD1 and HD2, also referred as HD), patient #249 and RSTS patient (CREBBPmut) (both referred as MUT); DEGs were selected based on nominal p value less than 0.05 and actual foldchange higher than 1.5 or lower than 0.67. (B) Volcano plot of DEGs between patients and controls, with log2 fold changes in gene expression on X-axis and the statistical significance (-log10 adjusted p value) on Y-axis; gene symbols of most significant DEGs (light blue) are displayed. (C) List of the top GO molecular functions enriched in DEGs resulted from transcriptomic analysis with respective adjusted p value

Fig 5: Schematic representation of HDAC2 pathogenetic variant found in patient #249. (A) Localization of HDAC2 variant (red line) at transcript (cDNA sequence with numbers referring to exons) and protein level (aa sequence) with different HDAC2 domains (homodimerization domain in green, histone deacetylase domain in orange, IACDE domain in yellow, coiled coil domain in purple). (B) Representation of wild type (wt) HDAC2 structure and protein with K444Lfs*61 predicted by SWISS-MODEL modelling server