Online Resource 1. The level of lipid peroxidation is elevated in HD brains. The total amount of lipid peroxidation (4-HNE plus MDA) was measured by a Colorimetric Microplate Assay.The experiment was carried out with five different sample sets (control, n=5 and HD, n=5) in triplicate and the stable chromophore was measured at 586nm of absorbance. The data was plotted relative to values obtained from standards. Data were analyzed by student-t test. *, Significantly different from control at p<0.05 (t=-2.35; DF=8).

Online Resource 2. Human brain samples that are used in the immunohistochemistry of 4-HNE. The grades of HD samples were between 2-3.

Control / Age / Gender
1 / 87 / F
2 / 61 / M
3 / 81 / M
4 / 81 / M
5 / 87 / F
Huntington’s disease
1 / 49 / M
2 / 72 / M
3 / 55 / M
4 / 80 / M
5 / 48 / M

Online Resource 3. Oxidative stress increases the cellular level of 4-HNE immunoreactivity in Tet-mtHtt-Q103-EGFP cells. Tet-mtHttQ103-EGFP SH-SY5Y cells were induced with 3 mM of doxycycline (Doxy) for 48 hr. Cells were treated with 10 M of H2O2 for 12 hr. The immunoreactivity of 4-HNE was determined by using the spinning disk confocal microscopy.

Online Resource 4. NDGA prevents neuronal death and DNA damage induced by oxidative stress in primary neurons. a, NDGA prevents neuronal death against metabolic oxidative stress induced by buthionine sulfoxide (BSO). NDGA is neuroprotective at 1 to 5 M concentration but the high dose (>10 M) shows no protection against oxidative stress. b. Phase contrast microscopy of primary neurons in the presence or absence of NDGA under oxidative stress condition. c, NDGA prevents oxidative stress-induced DNA damage in primary neurons.Neuronal DNA damage by BSO was detected by TUNEL labeling (green) in primary neurons (2 weeks in vitro). Differentiated cortical neurons were stained with III-tubulin (red) and the nucleus was counterstained with DAPI (blue). DNA damaged nuclei are shown as sky blue color in overlay panels. Nomarski views show the appearance of matured cortical neurons. TUNEL and immunofulorescence staining confirms that NDGA (1 M) protects oxidative stress-induced (BSO 2 mM) neuronal death.

Online Resource 5. NDGA prevents the loss of mitochondrial membrane potential (MPP) against glutamate (Glu)-induced neuronal cytotoxicity in primary cultures. Thisanalytical data was derived from the Figure 2 panel a. While Glu significantly reduced the MMP of primary neurons, pretreatment of NDGA restored the MMP of primary neurons close to the level of control (Cont). The MMP level was analyzed by NIH ImageJ program.

Online Resource 6. NDGA ameliorates mitochondrial membrane potential (MPP) in R6/2 HD mice. Thisanalytical data was derived from the Figure 6 panel a. The basal level of MMP was significantly reducedin R6/2 mice in comparison to WT (p<0.01). NDGA administration significantly improves the MMP in R6/2 mice (R6/2 + NDGA) in comparison to vehicle treated R6/2 mice (p<0.05). The MMP level was analyzed by NIH ImageJ program.

Online Resource 7. Modulation of mitochondrial ERE (MitoERE) reporter activity by NDGA. NDGA does not modulate MitoERE reporter activity in comparison to E2 (17beta-estradiol). Primary neurons were transiently transfected with pGL3E-MitoERE and pcDNA-ER constructs for 24 hr. Then NDGA (2 M) and E2 (2 M) were treated for 24 hr. Luciferase activity was normalized to the protein concentration of each sample. Oligonucleotides for human MitoERE (GCTTATAAACTTGACCAGGATCCAAGCTTA-TAAACTTGACCAGGA) was synthesized and subcloned to the pGL3E reporter vector.

Online Resource 8. 12-HETE, a byproduct of 12-lipoxygenase, prevents neuronal cell death induced by oxidative stress. Primary neurons were treated with indicated concentration of 12-HETE 1 hr before the treatment of homocysteic acid (HCA) (1 mM). 12-HETE (>100 nM) is neuroprotective against metabolic oxidative stress induced by HCA. Neuronal viability was measured by MTT assay.

Online Resource 9. The characterization of malondialdehyde (MDA), a maker of lipid peroxidation, immunoreactivity in R6/2 mice. a, Confocal images of mtHtt and MDA in WT, R6/2 and NDGA treated R6/2.Confocal microscopy was performed to detect the immunoreactivity of MDA. b, The level of MDA (pixel density) was slightly increased in R6/2 mice in comparison to control mice, but it was not significant at 9 weeks of age.

Supplementary Materials and Methods

Measurement of lipid peroxidation (malondialdehyde and 4-hydroxynoneal)

A quantitative estimation of lipid peroxidation (4-HNE plus MDA) in lysates from the striatal region of control and HD brains was carried out using a Colorimetric Microplate Assay for Lipid Peroxidation according to manufacturer's protocol (Oxford Biomedical Research Inc., Oxford, MI). This assay was based on the reaction of a chromogenic reagent, N-methyl-2-phenylindole (R1), with 4-HNE and MDA. The experiment was carried out with five different sample sets in triplicate and the stable chromophore was measured at 586nm of absorbance. The data was plotted relative to values obtained from standards.

Doxycyline-Inducible Q25 (wild type Htt)- and Q103 (mutant Htt)-EGFP Neuronal Cell Lines

The T-RExTM System (Invitrogen, Carlsbad, CA) was used to generate Q25 and Q103-EGFP cell lines. This system utilizes two vectors, the pcDNA6/TR vector, a regulatory plasmid that expresses the tetracycline repressor (TetR), and pcDNA4/TO that contains a CMV promoter driving the expression of the gene of interest under the control of Tet-operator sequences. Q25 and Q103-EGFP were subcloned into the pcDNA4/TO vector from pcDNA-Q25- and Q103-EGFP construct, in which exon 1 of mtHtt is cloned to an EGFP fusion vector (Clontech, Mountain View, CA). pcDNA4/TO-Q25- and Q103-EGFP were linearized and transfected into SH-SY5Y cell clone over expressing pcDNA6/TR. The Q25- and Q103-EGFP cell clones was selected by zeocin. For the induction of Q25- and Q103-EGFP, 3-10 M of doxycyline was treated into culture medium.

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Lee et al.