E grown in Iscove’s medium, supplemented with 10 (v/ v) FBS, 1 v/v non-essential amino acid solution, soybean purchase Mirin lipids, 0.1 (v/v) ITS-G, 3 (w/v) HSA, and 100 units/ml PEST (Gibco Cell Culture; Invitrogen, Sweden) at 37uC in a humidified atmosphere of 5 (v/v) CO2 in air. The cells were seeded at a density of 10,000 per well in 96-well microtiter plates (Falcon; Becton-Dickinson, NJ).Source of ReagentsHeparan, hyaluronic acid, chondroitin sulfate A, B, and C, catalase, and hydrogen peroxide (H2O2) were all purchased from Sigma-Aldrich, MedChemExpress Indolactam V Sweden.Binding of SAP to TTR in ELISABinding of SAP to pre-aggregated TTR was performed according to the protocol by [19] with some modifications.SAP and Aggregation-Induced Cell DeathDetermination of Cytotoxic ActivityThe cytotoxicity of TTR-A or TTR-D in the concentration range 0?0 mM and the effects of amyloid-associated molecules (0? mM) were assayed after 12 h in cultured IMR-32 cells using the WST-1 kit according to the manufacturer (Roche Diagnostics, Indianapolis, IN). The stable tetrazolium salt WST-1 (pinkcolored) is converted to a soluble formazan (orange-colored) by viable cells. Cells were incubated with the WST-1 reagent for 2 h at 37uC in a humidified atmosphere of 5 (v/v) CO2 in air. After this period, formation of the formazan dye was quantified as absorbance (at 450 nm for maximum formazan absorption and 690 nm as reference wavelength) with a scanning multiwell spectrophotometer (ELISA reader). All absorbance data were used first to calculate relative cell viability by dividing the absorbance of a sample by the absorbance of the control. Cells in medium without TTR aggregates or SAP served as controls, and their viability (equal to 1) was defined as no toxicity (0 ) according to the formula: (sample viability ?)6(21). All results are shown as the relative toxicity in relation to the controls and they are presented as mean values 6 SD. All experiments were performed in duplicate and were repeated at least three times.SAP expression (and no endogenous SAP) were used in the study (marked SAP-1, -3, -5, -9, -12, -16, -18, and -22 accordingly). The eye-specific driver w*; Pw+mC = GAL4-ninaE.GMR12, abbreviated GMR-Gal4 [61], was used and the UAS-TTR transgenic stocks have been described previously [32]. Expressing lines were generated using standard mating schemes. All flies were fed on standard mashed-potato/yeast/agar medium at 25uC and held under 12/12-h cycles of light and darkness.Assessment of Wing PhenotypeWing posture was assessed in female flies soon after eclosure by counting dragged wings in populations of at least 100 flies per genotype, in three independent experiments. Data are given as mean values 6 SD. Two independent UAS-TTR-A transgenic strains and nine independent UAS-SAP-transgenic strains were evaluated.Quantification of SAP Expression in Transgenic DrosophilaHeads of ten female flies were homogenized in 200 ml lysis buffer (50 mM Tris-HCl, pH 7.5, and 10 SDS (v/v) with complete protease inhibitors (Roche Diagnostics GmbH, Mannheim, Germany)) at 4uC, sonicated, boiled for 10 min at 100uC, and centrifuged for 10 min at 16,0006g (Eppendorf Microcentrifuge 5415C) at 4uC. Total protein concentrations in supernatants of fruit fly extracts were estimated with the BCA Protein Assay (PIERCE). Ten mg of total protein was resolved on 15 Criterion Tris-HCl precast gels (Bio-Rad Laboratories) and transferred electrophoretically onto Hybond-C Extra nitrocellulose membrane (0.45 m.E grown in Iscove’s medium, supplemented with 10 (v/ v) FBS, 1 v/v non-essential amino acid solution, soybean lipids, 0.1 (v/v) ITS-G, 3 (w/v) HSA, and 100 units/ml PEST (Gibco Cell Culture; Invitrogen, Sweden) at 37uC in a humidified atmosphere of 5 (v/v) CO2 in air. The cells were seeded at a density of 10,000 per well in 96-well microtiter plates (Falcon; Becton-Dickinson, NJ).Source of ReagentsHeparan, hyaluronic acid, chondroitin sulfate A, B, and C, catalase, and hydrogen peroxide (H2O2) were all purchased from Sigma-Aldrich, Sweden.Binding of SAP to TTR in ELISABinding of SAP to pre-aggregated TTR was performed according to the protocol by [19] with some modifications.SAP and Aggregation-Induced Cell DeathDetermination of Cytotoxic ActivityThe cytotoxicity of TTR-A or TTR-D in the concentration range 0?0 mM and the effects of amyloid-associated molecules (0? mM) were assayed after 12 h in cultured IMR-32 cells using the WST-1 kit according to the manufacturer (Roche Diagnostics, Indianapolis, IN). The stable tetrazolium salt WST-1 (pinkcolored) is converted to a soluble formazan (orange-colored) by viable cells. Cells were incubated with the WST-1 reagent for 2 h at 37uC in a humidified atmosphere of 5 (v/v) CO2 in air. After this period, formation of the formazan dye was quantified as absorbance (at 450 nm for maximum formazan absorption and 690 nm as reference wavelength) with a scanning multiwell spectrophotometer (ELISA reader). All absorbance data were used first to calculate relative cell viability by dividing the absorbance of a sample by the absorbance of the control. Cells in medium without TTR aggregates or SAP served as controls, and their viability (equal to 1) was defined as no toxicity (0 ) according to the formula: (sample viability ?)6(21). All results are shown as the relative toxicity in relation to the controls and they are presented as mean values 6 SD. All experiments were performed in duplicate and were repeated at least three times.SAP expression (and no endogenous SAP) were used in the study (marked SAP-1, -3, -5, -9, -12, -16, -18, and -22 accordingly). The eye-specific driver w*; Pw+mC = GAL4-ninaE.GMR12, abbreviated GMR-Gal4 [61], was used and the UAS-TTR transgenic stocks have been described previously [32]. Expressing lines were generated using standard mating schemes. All flies were fed on standard mashed-potato/yeast/agar medium at 25uC and held under 12/12-h cycles of light and darkness.Assessment of Wing PhenotypeWing posture was assessed in female flies soon after eclosure by counting dragged wings in populations of at least 100 flies per genotype, in three independent experiments. Data are given as mean values 6 SD. Two independent UAS-TTR-A transgenic strains and nine independent UAS-SAP-transgenic strains were evaluated.Quantification of SAP Expression in Transgenic DrosophilaHeads of ten female flies were homogenized in 200 ml lysis buffer (50 mM Tris-HCl, pH 7.5, and 10 SDS (v/v) with complete protease inhibitors (Roche Diagnostics GmbH, Mannheim, Germany)) at 4uC, sonicated, boiled for 10 min at 100uC, and centrifuged for 10 min at 16,0006g (Eppendorf Microcentrifuge 5415C) at 4uC. Total protein concentrations in supernatants of fruit fly extracts were estimated with the BCA Protein Assay (PIERCE). Ten mg of total protein was resolved on 15 Criterion Tris-HCl precast gels (Bio-Rad Laboratories) and transferred electrophoretically onto Hybond-C Extra nitrocellulose membrane (0.45 m.
