Fig. 7: The addition of GFP to the C-terminal part of Httex1 induces a differential structural organization as revealed by ICC and CLEM.

a Epitope mapping of the Httex1 antibodies. b Httex1 72Q-GFP inclusions formed 48 h after transfection in HEK cells were detected by ICC staining combined with confocal imaging. All Htt antibodies showed strong immunoreactivity to the periphery of the Httex1 inclusions and modest immunoreactivity to the core. The nucleus was counterstained with DAPI (blue) and the F-actin with phalloidin (red). Scale bars = 20 μm (left-hand panels) and 10 μm (middle and right-hand panels). c, d 48 h post-transfection, HEK cells were fixed, and ICC against Httex1 was performed and imaged by confocal microscopy (c). Scale bars = 5 μm. The selected area of the cells (white square) was then examined by EM (d). A binary image (inset) was generated from the electron micrograph using a median filtering and Otsu intensity threshold, allowing for a better distinction of the inclusions’ morphology. Scale bars = 500 nm. e Representative confocal images of Httex1 72Q-GFP nuclear inclusions formed in HEK cells 48 h after transfection. Httex1 expression (grey) was detected using a specific antibody against the N-terminal part of Htt (amino acids 1-17; 2B7 or Ab109115), and GFP (green) directly visualized in the appropriate channel. The nucleus was counterstained with DAPI (blue), and phalloidin (red) was used to stain filamentous actin. Httex1 nuclear inclusions are indicated by yellow arrowheads. Scale bars = 10 μm. f Electron micrograph of a representative Httex1 72Q-GFP inclusion. The white square indicates the area shown at higher magnification in the right-hand panel. The nucleus is highlighted in blue. Scale bars = 2 μm (left-hand panel) and 500 nm (right-hand panel).