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

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