construct encoding egfp  (Addgene inc)

Journal: bioRxiv

Article Title: A dual ribosomal system in the zebrafish soma and germline

doi: 10.1101/2024.08.29.610041

Figure Lengend Snippet: A) Schematic of the experimental strategy. eGFP-nanos3’UTR containing mRNA was injected into 1-cell stage zebrafish embryos from which two different experimental approaches were used to investigate the ratio of maternal and somatic ribosomes in PGCs and to test which ribosomes are bound to PGC-localized mRNAs. B) Brightfield and eGFP images of 1 day post-fertilization embryos, previously injected with only water, eGFP-nanos3’UTR mRNA, or biotinylated eGFP-nanos3-3’UTR mRNA. Scale bars indicate 500 μm. C) Ratio of maternal (yellow) versus somatic (blue) 18S and 28S rRNA in FACS-isolated eGFP-positive (PGCs) and eGFP-negative (somatic) cells analyzed by RT-qPCR. D) Ratio of maternal (yellow) versus somatic (blue) 18S and 28S variants associated with biotinylated eGFP-nanos3-3’UTR mRNAs isolated by RIP (RNA immunoprecipitation). Non-biotinylated eGFP-nanos3-3’UTR mRNA served as control. Non-specific (background) amplification was determined using IPs from wild-type (uninjected) lysate and was set to zero. In (C) and (D), three biological replicates from independent natural crosses were used, and significance was calculated by t-test (if not indicated, p-value > 0.05).

Article Snippet: To generate the RNA for microinjections, 2 μg of a construct encoding eGFP-nanos3’UTR was digested using NotI-HF (New England BioLabs, R3189S) for 1 h. The linearized product was purified according to manufacturer specifications via gel selection using NucleoSpin (Macherey-Nagel, 740588.50).

Techniques: Injection, Isolation, Quantitative RT-PCR, RNA Immunoprecipitation, Control, Amplification

Journal: bioRxiv

Article Title: VPS13C/PARK23 initiates lipid transfer and membrane remodeling for efficient lysosomal repair

doi: 10.1101/2025.10.23.684214

Figure Lengend Snippet: ( a ) U2OS wildtype (WT), VPS13C-KO1, VPS13C-KO2 or PI4K2A-KO cells were fed 3 μm polystyrene microbeads, treated with LLOMe (1 mM, 10 min) as indicated, immunostained for LAMP1 ( red ) and VAPA ( green ), and imaged by DeltaVision microscopy. Scale bar, 10 µm. ( b ) Time-lapse images of U2OS wildtype (WT), VPS13C-KO1, VPS13C-KO2 or PI4K2A-KO cells containing 3 µm pHrodo-labeled microbeads ( magenta ), expressing GFP-tagged OSBP ( green ) and treated with 1 mM LLOMe for the indicated time. Cells were imaged by spinning disk microscopy. Scale bar, 10 µm. ( c ) Time-lapse images of U2OS wildtype (WT), VPS13C-KO1 or PI4K2A-KO cells containing 3 µm pHrodo-labeled microbeads ( magenta ), expressing GFP-tagged PI4P sensor P4MX1 ( green ) and treated with 1 mM LLOMe for the indicated time. Cells were imaged by spinning disk microscopy. Scale bar, 10 µm. ( d ) Phase contrast (Phaco) and fluorescence images of polystyrene microbead-containing untreated or LLOMe-treated (1 mM, 10 min) U2OS wildtype (WT), VPS13C-KO1 or PI4K2A-KO cells immunostained for OSBP ( magenta ) and ALIX ( green ) or ORP9 ( magenta ) and hIST1 ( green ) and counterstained with DAPI ( blue ). Cells were imaged by spinning disk microscopy. Scale bar, 10 µm. ( e ) Percentage of microbead-containing lysosomes with positive immunostaining for OSBP or ORP9 in cells treated as in (d). Data are means ± SD of three independent experiments.

Article Snippet: The expression construct encoding eGFP-tagged P4Mx1 (Addgene, 108121) was described in Zewe et al. .

Techniques: Microscopy, Labeling, Expressing, Fluorescence, Immunostaining

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