Journal: Cellular and Molecular Life Sciences

Article Title: siRNA screening reveals that SNAP29 contributes to exosome release

doi: 10.1007/s00018-023-04822-8

Figure Lengend Snippet: Confocal microscopy analysis of SNAP29 in PC-3 cells. Control PC-3 cells ( A ) and cells depleted of SNAP29 ( B ) were fixed and permeabilized before incubation with SNAP29 antibody and the corresponding secondary antibodies. C–E Control cells showing SNAP29 labelling (green), Sec61A (ER) labelling (red), and both proteins together. F–H Control cells showing SNAP29 labelling (green), CD63 (MVBs) labelling (red), and both proteins together. I–K Control cells showing SNAP29 labelling (green), CellBrite (plasma membrane) staining (red), and a combination of both. L–N Control cells showing SNAP29 labelling (green), Rab5 (endosome) labelling (red), and both proteins together. In all cases, cells were washed and mounted with ProLong Gold antifade mounting medium containing DAPI to stain the nuclei (blue) and imaged using a Zeiss LSM710 laser scanning confocal microscope ( A–H ) or a Nikon ECLIPSE Ti2-E confocal spinning disk microscope ( I–N ). Images were captured with a × 63 objective ( A–H ) or a × 100 objective ( I–N ). Scale bars are indicated (10 μm)

Article Snippet: Then, cells were washed and mounted with ProLong Gold antifade mounting medium containing DAPI to stain the nuclei (blue) and imaged using a Zeiss LSM710 laser scanning confocal microscope or a Nikon ECLIPSE Ti2-E confocal spinning disk microscope.

Techniques: Confocal Microscopy, Incubation, Staining, Microscopy

Journal: Cellular and Molecular Life Sciences

Article Title: siRNA screening reveals that SNAP29 contributes to exosome release

doi: 10.1007/s00018-023-04822-8

Figure Lengend Snippet: Microscopy analysis of MVBs labelled for CD63 in control and SNAP29 depleted cells. Control PC-3 cells ( A ) and cells depleted of SNAP29 ( B ) were fixed and permeabilized before incubation with CD63 antibody and the corresponding secondary antibodies. Then, cells were washed and mounted with ProLong Gold antifade mounting medium containing DAPI to stain the nuclei (blue) and imaged using a Zeiss LSM710 laser scanning confocal microscope or a Nikon ECLIPSE Ti2-E confocal spinning disk microscope. Z-stack images were captured with a × 63 or × 100 objective. Scale bars are indicated (10 μm). C Quantification of the distance of CD63-positive puncta from the nucleus in control (mean = 3.26 nm) and SNAP29 depleted (mean = 2.12 nm) cells. The analysis of Z-stack images was performed by using the function Cell in IMARIS 9.0. At least 120 cells per condition, corresponding to more than 4500 puncta, were analyzed. Data shows mean ± SD. ***P < 0.0001. The quantification of the number of CD63 puncta, normalized to total cell area ( D ), puncta fluorescence intensity ( E ) and puncta area ( F ) was performed on Z-stack projections using ImageJ as described in Materials and Methods in control and SNAP29 depleted PC-3 cells. N ≥ 45 cells per condition or N ≥ 590 puncta per condition. Data shows mean ± SD. Cryo-sectioning and immuno-EM with CD63 (10 nm Au particles) in control cells showing ( G ) a classical MVB morphology and ( H ) an atypical MVB morphology containing multilaminar structures. Cryo-sectioning and immuno-EM with CD63 (10 nm) in SNAP29 depleted cells showing ( I ) classical MVB morphology and ( J ) an atypical MVB morphology containing multilaminar structures

Article Snippet: Then, cells were washed and mounted with ProLong Gold antifade mounting medium containing DAPI to stain the nuclei (blue) and imaged using a Zeiss LSM710 laser scanning confocal microscope or a Nikon ECLIPSE Ti2-E confocal spinning disk microscope.

Techniques: Microscopy, Incubation, Staining, Fluorescence

Journal: Cellular and Molecular Life Sciences

Article Title: siRNA screening reveals that SNAP29 contributes to exosome release

doi: 10.1007/s00018-023-04822-8

Figure Lengend Snippet: Analysis of MVB fusion events at the plasma membrane using CD63-pHluorin. CD63-pHluorin expressing PC-3 cell were imaged with a Nikon ECLIPSE Ti2-E confocal spinning disk microscope using a × 100 objective at or near the plasma membrane. Image analysis was performed using the ImageJ2/Fiji plugin ExoJ. A Total projection of fusion events (red circle) over a time course of 3 min in a representative cell. Scale bar, 10 μm. The white square depicts the region of interest enlarged in B . B Stills from live imaging of a fusion event (indicated by white arrows) over a time course of 33 s in a representative cell: before the event (top picture), at the start of the event (middle picture), and at the end (bottom picture). C Timelapse imaging (heat maps) of a representative CD63-pHluorin fusion event. Begin frame and peak frame are indicated. D Quantification of fusion events in control (mean = 2.00) and SNAP29 depleted (mean = 2.06) PC-3 cells transfected with CD63-pHluorin. N ≥ 18 cells per condition. E Fluorescent signal duration of CD63 fusion events in control (mean = 38.00 s) and SNAP29 depleted (mean = 32.77 s) cells. N ≥ 37 events per condition. F Delta intensity, i.e. the difference of fluorescence intensity between the begin frame and the peak frame, in control (mean = 524.6) and SNAP29 depleted (mean = 772.6) cells. N ≥ 37 events per condition. *P < 0.05; using Student’s two-tailed t test with Welch’s correction. For D – F , mean ± SD of two independent experiments is shown

Article Snippet: Then, cells were washed and mounted with ProLong Gold antifade mounting medium containing DAPI to stain the nuclei (blue) and imaged using a Zeiss LSM710 laser scanning confocal microscope or a Nikon ECLIPSE Ti2-E confocal spinning disk microscope.

Techniques: Expressing, Microscopy, Imaging, Transfection, Fluorescence, Two Tailed Test