Additional file 3: Figure S2. of Aβ accumulation causes MVB enlargement and is modelled by dominant negative VPS4A

(A) Confocal analysis of wt primary neurons show that untreated (DMSO) cells have no OC labelling, while cells incubated with Aβ1-40 for 48 h have low levels of OC labelling. However, cells incubated with Aβ1-42 display very strong OC labelling. (PDF 4679 kb

Additional file 1: Table S1. of Aβ accumulation causes MVB enlargement and is modelled by dominant negative VPS4A

List of antibodies. (PDF 35 kb

Additional file 6: Figure S5. of Aβ accumulation causes MVB enlargement and is modelled by dominant negative VPS4A

Aβ1-42 increases the diameter of LAMP-1 positive vesicles in N2a cells. Confocal images of exogenously added monomeric Aβ1-42 incubated for different time points, ranging from 15 min to 48 h, in N2a cells. 3D-rendering with Imaris from confocal z-stack. Colocalization of OC labelling and LAMP1 labelling can be seen from 45 min of Aβ treatment. The last image is from a single focal plane showing OC labelling inside an enlarged LAMP1-positive structure as well as OC labelling that appears to localize at the cell surface. Scale bar 10 μm. (PDF 375 kb

Additional file 10: Figure S9. of Aβ accumulation causes MVB enlargement and is modelled by dominant negative VPS4A

(A) 3D images show increased GSK3β labelling in dnVPS4-expressing N2a Swe compared to cells transfected with control plasmid. Rab7 labelling is also increased in dnVPS4 expressing cells. Scale bar 15 μm. (B) Western blot analysis of cell lysates of Swe N2a cells transfected with dnVPS4A showing no changes in total GSK3β or phosphorylated GSK3α/β (serine 21/9). (PDF 5570 kb

Additional file 9: Figure S8. of Aβ accumulation causes MVB enlargement and is modelled by dominant negative VPS4A

(A) Western blot analysis of APP and Aβ in Swe N2a cells treated with 5 nM bafilomycin A1 (BafA1) at different time points (h) before harvest. Cell culture media was replaced with fresh media 24 h before harvest. (B) Quantification of A. Values are normalized against actin and expressed as percentage of control, n = 3; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (ANOVA with Dunnett’s multiple comparisons test, compared to ctr). (C) Confocal images of 6E10 and LAMP1 labelling in Swe N2a cells treated with 5 nM bafilomycin A1 for the depicted times. At 24 h there is a build up of both 6E10 labelling and punctate LAMP1-positive structures. (D) Western blot analysis of APP and Aβ in Swe N2a cells treated with 5 nM bafilomycin A1 (BafA1) 24 h before harvest and 40 μg/ml cycloheximide (CHX) at different time points (h) before harvest. Cell culture media was replaced with fresh media 24 h before harvest, before the addition of Baf A1. (PDF 1310 kb

Fluorescently Guided Optical Photothermal Infrared Microspectroscopy for Protein-Specific Bioimaging at Subcellular Level

Infrared spectroscopic imaging is widely used for the visualization of biomolecule structures, and techniques such as optical photothermal infrared (OPTIR) microspectroscopy can achieve <500 nm spatial resolution. However, these approaches lack specificity for particular cell types and cell components and thus cannot be used as a stand-alone technique to assess their properties. Here, we have developed a novel tool, fluorescently guided optical photothermal infrared microspectroscopy, that simultaneously exploits epifluorescence imaging and OPTIR to perform fluorescently guided IR spectroscopic analysis. This novel approach exceeds the diffraction limit of infrared microscopy and allows structural analysis of specific proteins directly in tissue and single cells. Experiments described herein used epifluorescence to rapidly locate amyloid proteins in tissues or neuronal cultures, thus guiding OPTIR measurements to assess amyloid structures at the subcellular level. We believe that this new approach will be a valuable addition to infrared spectroscopy providing cellular specificity of measurements in complex systems for studies of structurally altered protein aggregates