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

Journal: bioRxiv

Article Title: A conformational switch driven by phosphorylation regulates Ykt6 activity in macroautophagy

doi: 10.1101/2020.03.15.992727

Figure Lengend Snippet: Ykt6 effects on autophagosome/lysosome fusion depend on the phosphorylation at the evolutionarily conserved site in the SNARE domain in the absence of endogenous Ykt6. (A) Representative western blot for LC3-II from a stably transfected PC12 cell line with a doxycycline inducible shRNA targeting endogenous rat Ykt6 and infected with lentiviruses carrying GFP-tagged wild-type (WT) human Ykt6 and phosphomutants. Cells were treated for 2 hours (2h) with 200nM Bafilomycin A 1 (Baf-A 1 ) in growing conditions (fresh 10% FBS and 4.5% glucose growth medium) or in starvation conditions (Torin-1 250nM, 1X HBSS supplemented with 10mM HEPES). Actin serves as a loading control. (B-C) Autophagic flux (defined as [LC3-II Baf-A 1 ] – [LC3-II]) from western blot in (A) of (B) growing and (C) starvation conditions. N=5 *p

Article Snippet: Autophagy was induced by treating the cells for 2 hours in 250nM Torin-1 (R & D Systems, 4247/10) in 1X Hanks’ Balanced salt solution, HBSS, with Calcium and Magnesium (Corning, 21-020-CV) supplemented with 10 mM HEPES buffer (Corning, 25-060-CI) at 37°C with 5% CO2 .

Techniques: Western Blot, Stable Transfection, Transfection, shRNA, Infection

Journal: bioRxiv

Article Title: A conformational switch driven by phosphorylation regulates Ykt6 activity in macroautophagy

doi: 10.1101/2020.03.15.992727

Figure Lengend Snippet: Ykt6 effects on autophagosome/lysosome fusion depend on the phosphorylation at the evolutionarily conserved site in the SNARE domain. Representative western blot for LC3-II from HEK293T transiently expressing GFP-tagged wild-type (WT) Ykt6 and phosphomutants. Cells were treated for 2 hours (2h) with 200nM Bafilomycin A 1 (Baf-A 1 ) in growing conditions (DMEM with 10% FBS and 4.5% glucose growth medium) or in starvation conditions (1X HBSS with 250nM Torin-1 and 10mM HEPES). Actin serves as a loading control. (B-C) Autophagic flux (defined as [LC3-II Baf-A 1 ] – [LC3-II]) from western blot in (A) of (B) growing and (C) starvation conditions. (A-C) N=6 *p

Article Snippet: Autophagy was induced by treating the cells for 2 hours in 250nM Torin-1 (R & D Systems, 4247/10) in 1X Hanks’ Balanced salt solution, HBSS, with Calcium and Magnesium (Corning, 21-020-CV) supplemented with 10 mM HEPES buffer (Corning, 25-060-CI) at 37°C with 5% CO2 .

Techniques: Western Blot, Expressing

Journal: bioRxiv

Article Title: A conformational switch driven by phosphorylation regulates Ykt6 activity in macroautophagy

doi: 10.1101/2020.03.15.992727

Figure Lengend Snippet: Ykt6 binding affinity for STX17, SNAP29 and Vamp8 is dependent on the phosphorylation at the evolutionarily conserved site in the SNARE domain. (A) Representative western blot of GFP immunoprecipitations from HEK293T cells expressing either GFP, wild-type (WT) GFP-Ykt6 or GFP-Ykt6 phosphomutants. Cells were treated for 2 hours (2h) in growing conditions (fresh 10% FBS and 4.5% glucose growth medium) or in starvation conditions (1X HBSS supplemented with 250nM Torin-1 and 10mM HEPES). (B) Quantification of STX17 and SNAP29 (A) relative to the efficiency of the pull down from each condition and normalized to WT Ykt6. N=3 *p

Article Snippet: Autophagy was induced by treating the cells for 2 hours in 250nM Torin-1 (R & D Systems, 4247/10) in 1X Hanks’ Balanced salt solution, HBSS, with Calcium and Magnesium (Corning, 21-020-CV) supplemented with 10 mM HEPES buffer (Corning, 25-060-CI) at 37°C with 5% CO2 .

Techniques: Binding Assay, Western Blot, Expressing