Journal: Bioscience Reports

Article Title: Amyloidogenic and non-amyloidogenic transthyretin variants interact differently with human cardiomyocytes: insights into early events of non-fibrillar tissue damage

doi: 10.1042/BSR20140155

Figure Lengend Snippet: Amyloidogenic V122I but not non-amyloidogenic T119M TTR induces the generation of superoxide species and caspase 3/7 activation in human cardiomyocytes ( a ) AC16 cells were treated with V122I TTR, T119M TTR, Antimycin A (positive control) or HBSS (vehicle control) for 24 h. The insults were then removed and the cells loaded with the superoxide-specific reagent DHE as detailed in the experimental section. Red fluorescence produced by DHE oxidation is shown. ( b ) AC16 cells were treated with V122I TTR, T119M TTR or, HBSS for 6 h at 37°C. Cell lysates were prepared as detailed in the experimental section and were added to Ac-DEVD-AFC caspase 3/7 substrate. Fluorescence intensity generated by cleavage of the substrate was measured. Asterisks in both panels indicate statistical significance with P

Article Snippet: When the recombinant TTR was intended to be used for biophysical studies, the gel filtration purification was performed in 10 mM phosphate buffer (sodium) pH 7.6/100 mM KCl/1 mM EDTA buffer (GF buffer); when the TTR was intended for cell culture experiments, HBSS (Hank's balanced salt solution; Mediatech) buffer was used instead.

Techniques: Activation Assay, Positive Control, Fluorescence, Produced, Generated

Journal: Bioscience Reports

Article Title: Amyloidogenic and non-amyloidogenic transthyretin variants interact differently with human cardiomyocytes: insights into early events of non-fibrillar tissue damage

doi: 10.1042/BSR20140155

Figure Lengend Snippet: Native V122I TTR competes with f-V122I TTR for the same interaction sites in human cardiomyocytes ( a ) AC16 cells were co-incubated with f-V122I TTR (320 nM) and increasing concentrations of amyloidogenic V122I TTR or non-amyloidogenic T119M TTR for 3 h at 4°C. The fluorescence associated with the cells was measured and normalized for total protein content. The data were normalized by the fluorescence from the samples incubated with f-V122I TTR only (100%). Filled triangles, V122I TTR; open circles, T119M TTR. ( b ) AC16 cells were co-incubated with f-V122I (600 nM) and equimolar amounts of unlabelled V122I TTR, T119M TTR, V122I stabilized with a small molecule (V122I-SM) or HBSS. Fluorescence associated with the cells was measured, normalized by total protein content and plotted as percentage with respect to the fluorescence of cells that had been incubated with f-V122I and HBSS only. The data show that similarly to T119M TTR, V122I-SM is unable to efficiently displace cell-associated f-V122I, whereas under the same conditions V122I TTR displaces more than 50% of f-V122I TTR.

Article Snippet: When the recombinant TTR was intended to be used for biophysical studies, the gel filtration purification was performed in 10 mM phosphate buffer (sodium) pH 7.6/100 mM KCl/1 mM EDTA buffer (GF buffer); when the TTR was intended for cell culture experiments, HBSS (Hank's balanced salt solution; Mediatech) buffer was used instead.

Techniques: Incubation, Fluorescence

Journal: Bioscience Reports

Article Title: Amyloidogenic and non-amyloidogenic transthyretin variants interact differently with human cardiomyocytes: insights into early events of non-fibrillar tissue damage

doi: 10.1042/BSR20140155

Figure Lengend Snippet: Differential interaction of the amyloidogenic V122I TTR and the non-amyloidogenic T119M TTR with the human cardiac cell line AC16 AC16 cells were treated with trypsin (or HBSS as control) to remove membrane-associated proteins. V122I and T119M TTR were then incubated with the cells for 4 h at 4°C or 37°C. Cell lysates were prepared and analysed by SDS—PAGE (15% gel) and Western blot using an anti-TTR and anti-actin antibodies. The lower panel shows densitometry analysis (absorbance) of total cell-associated TTR normalized to actin. Error bars represent S.D. of the biological duplicates. The percentage of TTR/actin ratio in cells pre-treated with trypsin (trypsin +) with respect to control cells (trypsin −) is shown.

Article Snippet: When the recombinant TTR was intended to be used for biophysical studies, the gel filtration purification was performed in 10 mM phosphate buffer (sodium) pH 7.6/100 mM KCl/1 mM EDTA buffer (GF buffer); when the TTR was intended for cell culture experiments, HBSS (Hank's balanced salt solution; Mediatech) buffer was used instead.

Techniques: Incubation, SDS Page, Western Blot