A rescue factor abolishing neuronal cell death by a wide spectrum of familial Alzheimer's disease genes and Abeta -- Data Supplement - Supplemental Data -- Proceedings of the National Academy of Sciences

Supplementary material for Hashimoto et al. (2001) Proc. Natl. Acad. Sci. USA 98 (11), 6336–6341. (10.1073/pnas.101133498)

Supplemental Figure 9 Fig. 9.
Lack of the effect of HN on neuronal cell death by Q79 and superoxide dismutase-1 (SOD1) mutants. (A) Lack of the effect of pHN or pHNG on neuronal cell death induced by EcD inducible expression of polyglutamine repeat Q79. F11/EcR cells were transfected with EcD-inducible Q79 plasmid (pDN-E/G5H-Q79) with an empty vector (pFLAG), pHN, or pHNG and then treated with (+) or without (-) 40 µM EcD for 72 hr. Cell mortality was measured by Trypan blue exclusion assay. All values indicate means ± SD of three independent transfections/treatments. (B) Lack of the effect of sHN, sHNG, or sHNA on neuronal cell death induced by EcD-inducible expression of Q79. F11/EcR cells were transfected with pDN E/G5H-Q79, treated with 1 µM sHN, 1 nM sHNG, or 1 µM sHNA, and then treated with (+) or without (-) EcD. The effect of 10 µM either polypeptide was similar. Cell mortality was similarly measured 72 hr after EcD treatment. (Inset) Cellular expression of induced Q79. Under the same condition as used for cell death assay, cell lysates were submitted to immunoblot analysis [1-5: HN polypeptide experiments in pDN-E/G5H-Q79-transfected cells (1: no EcD treatment; 2: 40 µM EcD treatment; 3: 40 µM EcD treatment + 10 µM sHN; 4: 40 µM EcD treatment + 10 µM sHNG; 5: 40 µM EcD treatment + 10 µM sHNA) and 6-8: pHN experiments in pDN-E/G5H-Q79-transfected cells (6: no EcD treatment + pFLAG transfection; 7: 40 µM EcD treatment + pFLAG transfection; 8: 40 µM EcD treatment + pHN transfection)]. (C) Lack of the effect of cotransfected pHN on neuronal cell death induced by expression of familial amyotrophic lateral sclerosis (FALS)-associated SOD1 mutants. F11 cells were transfected with pEF-BOS cording for FALS-associated mutant SOD1 (SOD1 with A4T, G85R, or G93A) with empty pFLAG or pHN. Cell mortality was similarly measured 72 hr after transfection. (D) Lack of the effect of sHN, sHNG, or sHNA on neuronal cell death induced by expression of FALS-associated SOD1 mutants. F11 cells were transfected with pEF-BOS coding for A4T, G85R, or G93A SOD1 in the presence or absence of 100 µM sHN, sHNG, or sHNA for 72 hr. Cell mortality was then measured by Trypan blue assay. (Inset) Cellular expression of transfected G85R-SOD1. Under the same condition as for cell death assay, cell lysates were submitted to immunoblot analysis [1-6: HN polypeptide experiments (1: no transfection; 2: pEF-BOS transfection; 3: G85R-SOD1 transfection; 4: G85R-SOD1 transfection + 10 µM sHN; 5: G85R-SOD1 transfection + 10 µM sHNG; 6: G85R-SOD1 transfection + 10 µM sHNA) and 7-10: pHN experiments (7: no transfection; 8: pEF-BOS + pFLAG transfection; 9: G85R-SOD1 + pFLAG transfection; 10: G85R-SOD1 + pHN transfection)].