Figure 5.

Differential nuclear localization of HDAC3 variants in HEK293T cells

(A) Immunofluorescence analysis of HEK293T cells transduced with wild-type (WT) or HDAC3 variants (p.Ala110Thr, p.Gly267Ser, p.Leu266Ser, and p.Arg359Cys). Cells were stained with an anti-FLAG antibody to visualize HDAC3 or its variants (green) and with DAPI to mark DNA (blue). HDAC3 WT prominently localizes in the nucleus, as shown by intense green fluorescence. In contrast, the p.Ala110Thr and p.Leu266Ser variants show notably weaker nuclear fluorescence and relatively stronger cytoplasmic fluorescence, implying compromised nuclear localization of HDAC3. In addition, the p.Gly267Ser variant exhibits relatively conserved nuclear localization similar to the WT, although the overall nuclear fluorescent intensity was weaker than the WT.

(B) Quantitative assessment of nuclear localization. This panel presents the quantification of nuclear versus cytoplasmic fluorescence intensity, expressed as the nuclear-to-cytoplasmic (N/C) ratio, in cells transduced with WT or variant forms of HDAC3. Individual data points correspond to measurements from single cells, with the median value of each variant depicted by a horizontal red line (cell counts of approximately 30–50 cells were used for each condition in the analysis). The N/C ratios for p.Ala110Thr, p.Leu266Ser, and p.Arg359Cys variants are reduced in comparison to the WT, indicating a deficiency in nuclear localization. Conversely, the p.Gly267Ser variant exhibits an N/C ratio similar to the WT, though the broad spread of data points indicates variable localization within the cell population. Statistical significance is indicated by asterisks (^∗∗∗∗p < 0.0001).