Journal: Traffic (Copenhagen, Denmark)
Article Title: Inhibition of Autophagy Alters Intracellular Transport of APP Resulting in Increased APP Processing.
doi: 10.1111/tra.12934
Figure Lengend Snippet: FIGURE 6 | Reduced localization of RUSH mCherry-APP-EGFP to LAMP1-positive vesicles. (A) ATG9 WT (left) and KO (right) HeLa cells stably expressing the Str-KDEL ER hook and mCherry-APP-EGFP were fixed after 60, 120, and 180 min of treatment with 40 μM biotin and were immunostained for the lysosome marker LAMP1 (magenta). (B) Co-occurrence analysis of LAMP1 and EGFP (left) or mCherry (right) signal was performed over the whole cell using automated thresholding and the image calculator operator “AND” in Fiji to generate an image with only co- occurring pixels. (C) Colocalization analysis of mCherry-APP-EGFP and LAMP1-positive structures between 0.01 and 1 μm2 in size was performed using the “analyze particles” function in Fiji. (D) Confocal microscopy images as shown in (A) were analyzed for LAMP1 vesicular structures. The left graph displays the number of LAMP1 vesicular particles per μm2, and the size of those particles is depicted in the right graph at 60, 120, and 180 min after releasing mCherry-APP-EGFP from the ER. (E) Co-occurrence analysis of mCherry and EGFP signal was conducted as described in (B). Statistical analysis was carried out using Mann–Whitney test on data from three independent experiments with n > 130 cells per condition (significance levels: *p < 0.05; **p < 0.01; ***p < 0.001).
Article Snippet: Due to insufficient ER retention of mCherry- APP- EGFP by the Ii- Str hook, the lentiviral plasmid pCDH_Str- KDEL (from Franck Perez, Addgene plasmid #65307) was used as another ER hook in conjunction with the cloned pLenti_CMV_SBPmCherry- APP- EGFP_blas vector.
Techniques: Stable Transfection, Expressing, Marker, Confocal Microscopy, MANN-WHITNEY
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