Released C-terminal HTT fragment elicits endoplasmic reticulum dilation, stress and cell death

1. Immunostainings of striatal cells transfected with mCherry-tagged C-HTT587-3144 fragment. Golgi apparatus compartments were immunostained with anti-GM130 (cis-Golgi), anti-CTR433 (medial-Golgi) and anti-TGN38 (trans-Golgi) antibodies. Early endosomes were either stained with anti-EEA1 or labelled with Rab5-GFP-expressing constructs. LC3-GFP-labelled autophagosomes and lysosomes were immunostained with anti-LAMP2.
2. Left: Striatal cells transfected with C-HTT587-3144 were treated with Z-DEVD-FMK, a caspase-3 inhibitor and analysed for cell death. Right: Non-transfected cells were treated for 2 h with 1 mM of H₂O₂ and Z-DEVD-FMK.
3. Toxicity in striatal cells transfected with C-HTT587-3144 and treated with Z-VAD-FMK.
4. Assessment of DNA fragmentation in C-HTT587-3144-expressing striatal cells using a TUNEL assay.
5. Toxicity in striatal cells transfected with C-HTT587-3144 fragment and siRNA targeted against Beclin-1 (two independent siRNA n1 & n2). Downregulation of Beclin-1 expression was assessed by immunoblotting.

Data information: The bar graphs (mean ± SEM) display pooled data from three to five independent experiments in triplicates. For the quantification of cell death after H₂O₂ treatment (B), one experiment was performed in duplicate and more than 1,000 cells were analysed. Statistics were done by unpaired t-test (B, C) or one-way ANOVA with Bonferroni’s multiple comparison test. ns: Non-significant, *P < 0.05.