plasmid plvx ef1a egfp rab7a ires puromycin  (Addgene inc)

Journal: The EMBO Journal

Article Title: BLTP3A is associated with membranes of the late endocytic pathway and is an effector of CASM

doi: 10.1038/s44318-025-00543-9

Figure Lengend Snippet: ( A ) Fluorescence images of RPE-1 cells expressing exogenous BLTP3A-mRFP (shown in inverted grays) and (not shown) GFP-tagged wild-type Rab7 (left) or dominant negative (DN) Rab7 (T22N) (right). Scale bar, 5 μm. ( B ) CLEM of a BLTP3A-mRFP positive cluster in an RPE-1 cell expressing GFP-tagged dominant negative Rab7. Left: fluorescence image of BLTP3A-mRFP (magenta). Scale bar, 1 μm. Right: EM micrograph of the field shown at left revealing that the BLTP3A-mRFP fluorescence reflects clusters of small vesicles. Scale bar, 500 nm. ( C ) BLTP3 chimeras design. Left: Surface representation of the predicted RBG core of BLTP3A. Red and blue indicate positive and negative charges, respectively, and gray indicates hydrophobic surfaces. Right: Surface representation (top) and ribbon representation (bottom) of the “untwisted” protein showing individual RBG motifs. Bottom: Cartoon of chimeras consisting of BLTP3A (dark orange) and BLTP3B (light orange) RBG motifs. ( D ) High-magnification live fluorescence images of RPE-1 cells expressing the indicated BLTP3-mRFP constructs (magenta) and LAMP1-GFP (green). Individual channels are shown as inverted grays. Scale bar, 1 μm. ( E ) Ribbon representation of the AlphaFold prediction of a.a. 1–336 of BLTP3A. Blue indicates loops connecting adjoining RBG motifs, and gray indicates the first beta-strand of the third RBG motif. ( F ) Fluorescence images (inverted grays) of RPE-1 cells expressing BLTP3A-1-336-mRFP and either (not shown) GFP-Rab7 (left), or GFP-Rab7 T22N (right). Scale bar, 5 μm. A zoom of an area of the cell at left (dotted square) expressing BLTP3A-1-336-mRFP (magenta) is also shown, along with the Rab7 fluorescence (green), demonstrating the localization of BLTP3A-1-336-mRFP around the entire profile of lysosomes. Individual channels are shown as inverted grays. Scale bar, 2 μm. ( G ) Cartoon depicting the proposed association of BLTP3A vesicle clusters with the surface of lysosomes and the dependence of this association on Rab7.

Article Snippet: GFP-Rab7 T22N , Addgene , RRID:Addgene_28048.

Techniques: Fluorescence, Expressing, Dominant Negative Mutation, Construct

Journal: bioRxiv

Article Title: MEG3 Enhances Survival of Developing Human Neurons with CLCN4 -Linked Autophagy Impairment

doi: 10.1101/2025.07.16.665078

Figure Lengend Snippet: (a) Representative images of day 6 neurons after Lysotracker treatment. Lysotracker is shown in magenta, and DAPI in blue. Scale bar = 20 μm. (b) Quantification of Lysotracker signal in day 6 neurons. The relative Lysotracker intensity of CLCN4 -variant neurons compared to the WT was presented. Each dot represents one biological replicate. (n=3-4, mean ± SEM, one-way ANOVA with Dunnett’s multiple comparisons test * p =0.039 for variant A; *** p =0.0007 for variant B; ** p =0.0092 for variant C) The magenta dashed line indicates the value for WT. (c) Relative acidity in acidic subcellular organelles. Each dot represents one biological replicate. (n=3, mean ± SEM, unpaired Student’s t-test; p =0.2894) (d-g) Dynamics of endo-lysosomal vesicles. RAB5, RAB7, RAB11 and LAMP1 indicates early endosome, late endosome, recycling endosome and lysosome markers, respectively. (RAB5, n=6; RAB7, n=3; RAB11, n=4; and LAMP1, n=3 or 4; mean ± SEM, unpaired Student’s t-test) (d) Representative images of a neuron with GFP-labeled vesicular organelles. TUJ1 and DAPI are shown in red and blue, respectively. Scale bar = 10 μm. (e) Relative GFP intensity expressed in vesicular organelles (* p =0.0265 for RAB5, * p =0.0158 for RAB7, * p =0.0475 for RAB11, and ** p =0.0067 for LAMP1) (f) Number of GFP-expressing vesicles in neurons. (* p =0.0186 for RAB5, * *p =0.0066 for RAB7, and p =0.9031 for LAMP1) (g) Average distance of GFP-expressing vesicles from the center of nucleus, representing the mean linear distance of puncta from the center of the nucleus in each cell. (** p =0.0011 for RAB5, * p =0.0463 for RAB7, and * p =0.0103 for LAMP1) (h) Scheme of autophagic flux analysis using tandem fluorescent protein fused to LC3. (i) Representative images of day 6 neurons expressing tandem fluorescent-tagged LC3. Red: RFP, green: GFP, white: TUJ1. Scale bar = 5 μm. (j) Quantification of the GFP-/RFP+ puncta. (n=5, mean ± SEM, unpaired Student’s t-test; * p =0.0164)

Article Snippet: For specific subcellular vesicle visualization, lentiviral constructs encoding GFP-tagged RAB5 (Addgene #134858), RAB7 (Addgene #133027), RAB11 (Addgene #134860), and LAMP1 (Addgene #134868) were utilized.

Techniques: Variant Assay, Labeling, Expressing