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: Immunology

Article Title: Regulation of CTLA‐4 recycling by LRBA and Rab11

doi: 10.1111/imm.13343

Figure Lengend Snippet: CTLA‐4 colocalizes with endosomal Rab GTPases. (a,b) CTLA‐4‐transduced HeLa (a) or Jurkat (b) cells were fixed, permeabilized, and stained with human anti‐CTLA‐4 Ab and rabbit anti‐Rab5, anti‐Rab7, anti‐Rab9 or anti‐Rab11 Abs followed by goat anti‐human IgG‐Alexa Fluor 546, donkey anti‐rabbit IgG‐Alexa Fluor 488, Hoechst and CTV. Cells were analysed by confocal microscopy. Scale bars = 10 μm. (c) Graph showing quantification of the colocalization of CTLA‐4 vesicles with Rab vesicles in (a) or (b). Data shown as mean ± SEM, n = 3. *** P < 0.005 determined by two‐way ANOVA with Sidak's multiple comparisons test

Article Snippet: The following Rab‐GFP constructs were acquired from Addgene and generated by Marci Scidmore (Rzomp et al; Cortes et al) (Rab4 WT, DN, CA; Rab5 WT; Rab11 CA), Sergio Grinstein (Bohdanowicz et al) (Rab5 DN, CA) and Richard Pagano (Choudhury et al) (Rab9 WT, DN; Rab11 WT, DN): Rab4WT (Addgene plasmid #49434), Rab4 DN (#49476), Rab4 CA (#49475), Rab5 WT (#49888), Rab5 DN (#35141), Rab5 CA (#35140), Rab9 WT (#12663), Rab9 DN (#12664), Rab11 WT (# 12674), Rab11 DN (#12678) and Rab11 CA (#49553).

Techniques: Staining, Confocal Microscopy

Journal: Immunology

Article Title: Regulation of CTLA‐4 recycling by LRBA and Rab11

doi: 10.1111/imm.13343

Figure Lengend Snippet: CTLA‐4 surface expression is regulated by Rab5 and Rab11. CTLA‐4‐transduced HeLa or Jurkat cells were transfected with Rab‐GFP constructs (empty vector—EV; wild type—WT; dominant negative—DN; or constitutively active—CA) for 24 h, and then stained for surface CTLA‐4 expression. (a) Flow cytometry contour plots of HeLa cells showing Rab‐GFP vs surface CTLA‐4 expression. (b) Flow cytometry plot showing the GFP + gate used. (c,d) Graphs showing surface CTLA‐4 expression relative to empty vector control in GFP + gated HeLa (c) or Jurkat (d) cells. Data shown as mean ± SD, c: n ≥ 3, d: n ≥ 4. Differences between conditions and EV determined by one‐way ANOVA and Dunnett's multiple comparisons test; * P < 0.05, *** P < 0.005 and **** P < 0.001

Article Snippet: The following Rab‐GFP constructs were acquired from Addgene and generated by Marci Scidmore (Rzomp et al; Cortes et al) (Rab4 WT, DN, CA; Rab5 WT; Rab11 CA), Sergio Grinstein (Bohdanowicz et al) (Rab5 DN, CA) and Richard Pagano (Choudhury et al) (Rab9 WT, DN; Rab11 WT, DN): Rab4WT (Addgene plasmid #49434), Rab4 DN (#49476), Rab4 CA (#49475), Rab5 WT (#49888), Rab5 DN (#35141), Rab5 CA (#35140), Rab9 WT (#12663), Rab9 DN (#12664), Rab11 WT (# 12674), Rab11 DN (#12678) and Rab11 CA (#49553).

Techniques: Expressing, Transfection, Construct, Plasmid Preparation, Dominant Negative Mutation, Staining, Flow Cytometry

Journal: Immunology

Article Title: Regulation of CTLA‐4 recycling by LRBA and Rab11

doi: 10.1111/imm.13343

Figure Lengend Snippet: CTLA‐4 degradation is regulated by Rab5 and Rab7. CTLA‐4‐transduced HeLa or Jurkat cells were transfected with Rab‐GFP constructs (empty vector—EV; wild type—WT; dominant negative—DN; or constitutively active—CA) for 24 h, and then stained with anti‐CTLA‐4 PE at 37°C for 1 h. Cells were then incubated at 37°C for up to 4 h, before being fixed, and analysed by flow cytometry. (a,c) Flow cytometry histogram of HeLa (a) or Jurkat (c) cells showing GFP + gate used for analysis. (b,d) Graphs showing CTLA‐4 staining in GFP + gated HeLa (b) or Jurkat (d) cells, relative to 0 h. Data shown as mean ± SD, n ≥ 3. Differences between conditions were determined by two‐way ANOVA and Dunnett's multiple comparisons test; * P < 0.05, ** P < 0.01 and **** P < 0.001, ns—non‐significant

Article Snippet: The following Rab‐GFP constructs were acquired from Addgene and generated by Marci Scidmore (Rzomp et al; Cortes et al) (Rab4 WT, DN, CA; Rab5 WT; Rab11 CA), Sergio Grinstein (Bohdanowicz et al) (Rab5 DN, CA) and Richard Pagano (Choudhury et al) (Rab9 WT, DN; Rab11 WT, DN): Rab4WT (Addgene plasmid #49434), Rab4 DN (#49476), Rab4 CA (#49475), Rab5 WT (#49888), Rab5 DN (#35141), Rab5 CA (#35140), Rab9 WT (#12663), Rab9 DN (#12664), Rab11 WT (# 12674), Rab11 DN (#12678) and Rab11 CA (#49553).

Techniques: Transfection, Construct, Plasmid Preparation, Dominant Negative Mutation, Staining, Incubation, Flow Cytometry