Journal: Human mutation

Article Title: Biallelic loss of function variants in PPP1R21 cause a neurodevelopmental syndrome with impaired endocytic function.

doi: 10.1002/humu.23694

Figure Lengend Snippet: FIGURE 2 PPP1R21 co-localizes with the main early endosome protein EEA1 but not Golgi proteins and PPP1R21 staining is absent in fibroblasts obtained from patient 3_V:4 who is homozygous for c.1607dupT p.(Leu536Phefs*7). (A) In order to determine the entity of PPP1R21 positive vesicles, we performed co-localization studies using Golgi marker GM130 (mouse, ab169276, Abcam, USA) and 58K (ab27043 mouse, Abcam, USA) as well as EEA1 antibody to mark the early endosome. While no co-localization with Golgi markers was observed (upper panels), PPP1R21 (rabbit, HPA036792, Atlas antibodies, Sweden) nearly completely co-localized with EEA1 (lower panel). Scale bar 20 𝜇m. (B) Using two different PPP1R21 antibodies, HPA036792 (ab1, antibody epitope is represented by aminoacid aa161-256) and HPA 036791 (ab2, antibody epitope is represented by aminoacid aa572-666), we found complete loss of the vesicular staining pattern in PPP1R21 mutant fibroblasts we previously observed in control cells while no difference regarding EEA1 staining was detected between PPP1R21 mutant and control fibroblasts. Scale bar: 20 𝜇m

Article Snippet: See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense REHMAN ET AL. 277 F IGURE 2 PPP1R21 co-localizes with themain early endosome protein EEA1 but not Golgi proteins and PPP1R21 staining is absent in fibroblasts obtained from patient 3_V:4 who is homozygous for c.1607dupT p.(Leu536Phefs*7). (A) In order to determine the entity of PPP1R21 positive vesicles, we performed co-localization studies using Golgi marker GM130 (mouse, ab169276, Abcam, USA) and 58K (ab27043mouse, Abcam, USA) as well as EEA1 antibody tomark the early endosome.While no co-localization with Golgi markers was observed (upper panels), PPP1R21 (rabbit, HPA036792, Atlas antibodies, Sweden) nearly completely co-localized with EEA1 (lower panel).

Techniques: Staining, Marker, Mutagenesis, Control

Journal: bioRxiv

Article Title: Early endosome disturbance and endolysosomal pathway dysfunction in Duchenne muscular dystrophy

doi: 10.1101/2024.12.16.628552

Figure Lengend Snippet: Representative confocal images of immortalized primary DMDΔ45-52, DMDΔ52 and healthy controls (Ctrl) (A) myoblasts and (D) myotubes stained with DAPI to mark nuclei (blue) and anti-EEA1 antibody to mark early endosomes (green). Quantification of EEA1 positive puncta normalized by cell area on confocal microscopy images showed an increased number of EEA1 positive puncta in DMD myoblasts (B) and myotubes (E) compared to Ctrl. The data are represented as the mean ± SEM of EEA1 puncta number per cell area of at least 88 cells per condition (n = 3 independent experiments). Quantification of EEA1 puncta mean size (µm 2 ) from confocal microscopy images of (C) DMD myoblasts and ( F) myotubes compared to their respective Ctrl. The data are represented as the mean ± SEM of EEA1 puncta size per cell of at least 100 cells per condition (n = 3 independent experiments). Scale bar = 10µm. Statistics: one-way ANOVA with a post hoc Bonferroni test, **p<0.01, ****p<0.0001.

Article Snippet: Cells were then incubated with a goat monoclonal antibody EEA1 (1/500; SICGEN, Cantanhede, Portugal), a rabbit monoclonal antibody Rab7 (1/200; Santa Cruz Biotechnology, Heidelberg, Germany) and a mouse monoclonal antibody LAMP1 (1/100; Santa Cruz Biotechnology, Heidelberg, Germany) in PBS supplemented with 0.5% Triton X-100 for 2h at room temperature, washed with PBS and incubated with the appropriate secondary antibodies labeled with Alexa Fluor 488 or Alexa Fluor 568 (1/1000; Life Technologies, Villebon-sur-Yvette, France) for 45 min at RT and, with 4’,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich Chimie, Saint Quentin Fallavier, France) for 5 min for nuclei staining.

Techniques: Staining, Confocal Microscopy

Journal: bioRxiv

Article Title: Early endosome disturbance and endolysosomal pathway dysfunction in Duchenne muscular dystrophy

doi: 10.1101/2024.12.16.628552

Figure Lengend Snippet: Low and high magnification electron micrographs of early endosomes (EEs) in myoblasts from (A) healthy controls (Ctrl) and (B) DMDΔ45-52 patients after EEA1 immunogold-labeling showing large isolated EEs (> 1µm, asterisk) and clusters (rectangular outlines) of at least 2 endosomes that are more frequently observed in DMD myoblasts compared to controls (Ctrl). Early endosomes are colored in purple for clarity.

Article Snippet: Cells were then incubated with a goat monoclonal antibody EEA1 (1/500; SICGEN, Cantanhede, Portugal), a rabbit monoclonal antibody Rab7 (1/200; Santa Cruz Biotechnology, Heidelberg, Germany) and a mouse monoclonal antibody LAMP1 (1/100; Santa Cruz Biotechnology, Heidelberg, Germany) in PBS supplemented with 0.5% Triton X-100 for 2h at room temperature, washed with PBS and incubated with the appropriate secondary antibodies labeled with Alexa Fluor 488 or Alexa Fluor 568 (1/1000; Life Technologies, Villebon-sur-Yvette, France) for 45 min at RT and, with 4’,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich Chimie, Saint Quentin Fallavier, France) for 5 min for nuclei staining.

Techniques: Labeling, Isolation

Journal: bioRxiv

Article Title: Early endosome disturbance and endolysosomal pathway dysfunction in Duchenne muscular dystrophy

doi: 10.1101/2024.12.16.628552

Figure Lengend Snippet: Representative confocal images showing sections of muscle biopsies from (A) 3-, 5- and 10-year-old DMD patients (respectively DMDΔ45-52, DMDΔ52, DMDΔ48-50) and age-matched healthy controls (n=1 for each) and (B) severely (S) (n=3) or moderately (M) (n=3) affected GRMD dogs of 6 month-old (6 m) and age-matched healthy control dogs (n=3) stained with DAPI to mark nuclei, anti-caveolin (for dog) or anti-laminin alpha-2 (for human) antibodies to mark the plasma membrane (white) and EEA1 antibody to mark early endosomes (green) or (E) myofibers isolated from the EDL muscle of wild type (WT) and mdx of 12 week-old (n=4) labeled with DAPI to mark nuclei (blue), anti-tubulin antibody to mark the microtubule cytoskeleton (green), and anti-EEA1 antibody to mark EEs (red). Quantification of EEA1 positive puncta showed a significant increase in early endosome staining in muscles of (C) GRMD of 6-month-old (6 m, n=3) and (D) 2-month-old (2 m, n=3) and, (F) 12 week-old (12 w, n=4) and (G) 2 week-old (2 w, n=3) mdx fibers compared to their respective controls. The data are represented as the mean ± SEM of at least 500 fibers per dog and 15 to 26 myofibers per group of mice analyzed on confocal images. Statistics: one-way ANOVA with a post hoc Bonferroni test, for human and dog analysis and unpaired Student’s t-test (two tailed) for mice. **p<0.01, ***p<0.001, ****p<0.0001, ns: non-significant.

Article Snippet: Cells were then incubated with a goat monoclonal antibody EEA1 (1/500; SICGEN, Cantanhede, Portugal), a rabbit monoclonal antibody Rab7 (1/200; Santa Cruz Biotechnology, Heidelberg, Germany) and a mouse monoclonal antibody LAMP1 (1/100; Santa Cruz Biotechnology, Heidelberg, Germany) in PBS supplemented with 0.5% Triton X-100 for 2h at room temperature, washed with PBS and incubated with the appropriate secondary antibodies labeled with Alexa Fluor 488 or Alexa Fluor 568 (1/1000; Life Technologies, Villebon-sur-Yvette, France) for 45 min at RT and, with 4’,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich Chimie, Saint Quentin Fallavier, France) for 5 min for nuclei staining.

Techniques: Control, Staining, Membrane, Isolation, Labeling, Muscles, Two Tailed Test

Journal: bioRxiv

Article Title: Early endosome disturbance and endolysosomal pathway dysfunction in Duchenne muscular dystrophy

doi: 10.1101/2024.12.16.628552

Figure Lengend Snippet: The expression of Rab5 mRNA analyzed by RT-PCR (top) and Rab5 protein analyzed by western-blot (bottom) in (A) DMD myoblasts (n = 3 independent experiments) (B) dog muscles (n = 5 biopsies from 5 WT dogs and n=5 biopsies from 4 GRMD dogs) and (C) mdx mouse muscles (n = 4 mice per group). RT-qPCR were performed in duplicate. The data are represented as the mean ± SEM. Statistics: one-way ANOVA with a post hoc Bonferroni test for (A) DMD myoblasts comparison and unpaired Student t-test (two tailed) for (B) dog and (C) mice comparisons. (D) Western-blot analysis and quantification (n=3) of Rab5 protein showing the efficacy of Rab5 silencing in human DMDΔ45-52 myoblasts transfected with siRNA directed against Rab5 (siRab5) compared to DMD and control myoblasts (Ctrl) treated with a scrambled siRNA control (siScr). (E) The number of EEA1 positive endosomes is reduced in human DMD cells treated with siRab5, to a level equivalent to that of control cells (Ctrl). The data are represented as the mean ± SEM of 3 independent experiments for western-blot and at least 300 cells of three independent experiments analyzed on confocal images for early endosome counting. Statistics: one-way ANOVA with a post hoc Bonferroni test. *p<0.05, **p<0.01, ***p<0.01, ****p<0.0001. ns: non-significant.

Article Snippet: Cells were then incubated with a goat monoclonal antibody EEA1 (1/500; SICGEN, Cantanhede, Portugal), a rabbit monoclonal antibody Rab7 (1/200; Santa Cruz Biotechnology, Heidelberg, Germany) and a mouse monoclonal antibody LAMP1 (1/100; Santa Cruz Biotechnology, Heidelberg, Germany) in PBS supplemented with 0.5% Triton X-100 for 2h at room temperature, washed with PBS and incubated with the appropriate secondary antibodies labeled with Alexa Fluor 488 or Alexa Fluor 568 (1/1000; Life Technologies, Villebon-sur-Yvette, France) for 45 min at RT and, with 4’,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich Chimie, Saint Quentin Fallavier, France) for 5 min for nuclei staining.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Muscles, Quantitative RT-PCR, Comparison, Two Tailed Test, Transfection, Control

Journal: bioRxiv

Article Title: Early endosome disturbance and endolysosomal pathway dysfunction in Duchenne muscular dystrophy

doi: 10.1101/2024.12.16.628552

Figure Lengend Snippet: (A) The expression of Rab5 mRNA analyzed by RT-PCR (left) and Rab5 protein analyzed by western blot (right) in muscles of WT dogs (n=5 for mRNA or n=1 for protein), GRMD (n= 5 dogs for mRNA or n=2 dogs for protein), AAV-U7snRNA treated GRMD to restore dystrophin (n = 7 biopsies from 3 treated GRMD for mRNA, NaCl-treated controls (NaCl-GRMD, n=1), or n=2 biopsies per dog from 2 treated GRMD (for protein) showing that expression of Rab5 is decreased by dystrophin restoration in treated GRMD. Actin was used as a loading buffer in western blot. (B) Representative sections of muscle biopsies isolated from moderately affected GRMD and AAV-U7snRNA treated GRMD labeled with DAPI to mark nuclei (blue), anti-caveolin antibody to mark the plasma membrane (white) and anti-EEA1 antibody to mark early endosomes (green). Quantification of EEA1 positive puncta showed a significant decrease in early endosome staining in GRMD treated with an AAV-U7snRNA (n=2 biopsies from 1 treated GRMD) compared to NaCl-treated controls (NaCl-GRMD, n=1) showing that the therapeutically restored dystrophin allowed a partial restoration of early endosomes number compared to controls. The data are represented as the mean ± SEM of at least 500 fibers per dog analyzed on confocal images. Sale Bar = 50 µm. Statistics: one-way ANOVA with a post hoc Bonferroni test for panel A (Rab5 mRNA) and unpaired Student’s t-test (two tailed) for panel B (EEs quantification), *p<0.05; ****p<0.0001, ns: non-significant.

Article Snippet: Cells were then incubated with a goat monoclonal antibody EEA1 (1/500; SICGEN, Cantanhede, Portugal), a rabbit monoclonal antibody Rab7 (1/200; Santa Cruz Biotechnology, Heidelberg, Germany) and a mouse monoclonal antibody LAMP1 (1/100; Santa Cruz Biotechnology, Heidelberg, Germany) in PBS supplemented with 0.5% Triton X-100 for 2h at room temperature, washed with PBS and incubated with the appropriate secondary antibodies labeled with Alexa Fluor 488 or Alexa Fluor 568 (1/1000; Life Technologies, Villebon-sur-Yvette, France) for 45 min at RT and, with 4’,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich Chimie, Saint Quentin Fallavier, France) for 5 min for nuclei staining.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Muscles, Isolation, Labeling, Membrane, Staining, Two Tailed Test