Journal: Frontiers in Genetics

Article Title: Down Syndrome Fetal Fibroblasts Display Alterations of Endosomal Trafficking Possibly due to SYNJ1 Overexpression

doi: 10.3389/fgene.2022.867989

Figure Lengend Snippet: The dynamics of early endosomes is perturbed in DS-HFFs. (A) EU-HFFs and DS-HFFs were stained with Rab5 antibody detected with Alexa-546-conjugated secondary antibodies. Serial confocal sections were collected from the top to the bottom of the cells. For each condition, the 3D reconstruction and corresponding intensity map are shown. Scale bars, 8 μm. Mean fluorescence intensity (arbitrary unit, a.u.) of Rab5-positive structures is higher in DS-HFFs than in EU-HFFs. The bars show the relative mean value ± SD of three independent experiments. (B) Representative 3D reconstruction and corresponding intensity map of cell ROIs taken into analysis (left panels). Two populations of Rab5 endosomes are distinguishable in terms of brightness, with lower (arrows) and higher (arrowheads) intensity fluorescent signal. Representative scatter dot plot showing the distribution of fluorescence intensity of Rab5-positive structures in EU-HFFs and DS-HFFs (right panel). Each dot represents one sample value, error bars represent mean value ± SD. (C) The percentage of larger Rab5 positive structures (>1 μm) are increased in DS-HFFs when compared to that in EU-HFFs. The bars show the relative mean value ± SD of three independent experiments, n ≥ 30 cells. (D) Representative immunoblotting of Rab5 in DS-HFFs and EU-HFFs. GAPDH was used as the loading control. The molecular weight of protein markers is indicated. Densitometric analysis of three different experiments, performed in three euploid and in four trisomic cultures, is shown. Error bars, mean ± SD. * p < 0.05; *** p < 0.001, Student’s t -test.

Article Snippet: Primary antibodies included the following: rabbit polyclonal anti-Cathepsin D (Thermo Fisher Scientific), rabbit polyclonal anti-EEA1 (Thermo Fisher Scientific), rabbit polyclonal anti-FGF receptor 1 (Cell Signaling Technology), mouse monoclonal anti-Lamp-1 (BD Pharmingen™), mouse monoclonal anti-Rab5 (BD Transduction Laboratories™), mouse monoclonal anti-Rab7 (Santa Cruz Biotechnology, Inc.), and rabbit polyclonal anti-Synj1 (Abcam plc).

Techniques: Staining, Fluorescence, Western Blot, Molecular Weight

Journal: British Journal of Pharmacology

Article Title: High glucose‐mediated PICALM and mTORC1 modulate processing of amyloid precursor protein via endosomal abnormalities

doi: 10.1111/bph.15131

Figure Lengend Snippet: High glucose up‐regulates Aβ production through early endosomal enlargement. (a–c) Samples were obtained from ZLC and ZDF rat hippocampus. (a) C99, Rab5, and β‐actin were detected by western blot. n = 5 in each group. (b) Aβ 1–42 were measured by rat and mouse Aβ 1–42 specific ELISA assay. n = 6 in each group with two technical replicates each. (c) Tissue slides for IHC were immunostained with Rab5‐specific antibody and counterstained with DAPI. Scale bars, 50 μm (magnification, ×200). n = 5 in each group. * P < .05, significantly different from ZLC rats. (d,e) Samples were obtained from vehicle‐ and STZ‐treated mice hippocampus. (d) C99, Rab5, and β‐actin were detected by western blot. n = 5 in each group. (e) Tissue slides for IHC were immunostained with Rab5‐specific antibody and counterstained with DAPI. Scale bars, 200 μm (magnification, ×200). n = 5 in each group. *P < .05, significantly different from vehicle‐treated mice. (f) The SK‐N‐MC cells were treated with d‐glucose (25 mM) or l‐glucose (25 mM) for 24 h. Then, C99, Rab5, and β‐actin were detected by western blot. n = 5 from independent experiments. (g) d‐glucose (25 mM) or l‐glucose (25 mM) was treated for 48 h in the cells. Then, Aβ 1–42 from cell culture medium were measured by human Aβ 1–42 specific ELISA assay. n = 5 from independent experiments with two technical replicates each. (h) The cells were treated with high glucose (25 mM) for 24 h. Active Rab5 (GTP‐bound Rab5) was immunoprecipitated with Rab5‐specific antibody (upper panels). Expression of Rab5 and β‐actin in total lysate is shown in the middle and lower panels. n = 5 from independent experiments. (i) The cells were treated with high glucose (25 mM) for 24 h which were immunostained with Rab5 and BACE1‐specific antibodies and counterstained with DAPI. Scale bars, 8 μm (magnification, ×1,000). n = 7 from independent experiments. (j) Immunostaining of cells treated with high glucose (25 mM) for 24 h was visualized. Rab5 and APP (C‐terminus)‐specific antibodies were used and counterstained with DAPI. Scale bars, 8 μm (magnification, ×1,000). n = 6 from independent experiments. *P < .05, significantly different from control. (k,l) The cells were transfected with NT siRNA or RAB5 siRNA for 12 h prior to high glucose (25 mM) treatment for 24 and 48 h, respectively. (k) C99 and β‐actin were detected by western blot. n = 5 from independent experiments. (l) Aβ 1–42 from cell culture medium were measured by human Aβ 1–42 specific ELISA assay. n = 5 from independent experiments with two technical replicates each. Logarithmic transformations were performed for homogeneity of the sample variance. * P < .05, significantly different from NT siRNA transfection, # P < .05, significantly different from high glucose with NT siRNA transfection. Quantitative data are presented as a mean ± SEM. All blots and immunofluorescence images are representative

Article Snippet: Briefly, cells were incubated with the provided lysis buffer containing anti‐active Rab5 monoclonal antibody (NewEast Biosciences, #26911, RRID:AB_2617182).

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay, Cell Culture, Immunoprecipitation, Expressing, Immunostaining, Transfection, Immunofluorescence

Journal: British Journal of Pharmacology

Article Title: High glucose‐mediated PICALM and mTORC1 modulate processing of amyloid precursor protein via endosomal abnormalities

doi: 10.1111/bph.15131

Figure Lengend Snippet: High glucose facilitates lipid raft‐mediated APP endocytosis resulting in early endosomal enlargement. (a) Biotin surface labelling and internalization assay from the SK‐N‐MCs treated with high glucose (25 mM) for 24 h were carried out. 0 and 15 min indicate time points of internalization. In the bottom, total lysates were subjected to western blot. APP and β‐actin were detected. Percent of internalized APP was measured by normalizing with total APP at time 15 in each conditions. n = 5 from independent experiments. (b) High glucose (25 mM) was treated for 24 h in the cells. Sucrose gradient‐fractionized lysates were subjected to western blot. APP, CAV1, and FLOT1 were detected. Exploratory data. (c,d) The cells were treated with GM6001 (20 μM) for 30 min and then with MβCD (1 mM) for 30 min and high glucose (25 mM) for 24 h. (c) Cell surface was biotinylated, and labelled proteins were pulled down by streptavidin beads. Surface APP, total APP, and β‐actin were detected by western blot. n = 5 from independent experiments. (d) Biotin surface labelling and internalization assay including 15 min for internalization were carried out. Internalized APP, total APP, and β‐actin were detected by western blot. n = 5 from independent experiments. Logarithmic transformations were performed for homogeneity of the sample variance. (e) The cells were incubated with MβCD (1 mM) for 30 min prior to high glucose treatment (25 mM) for 24 h. The cells were immunostained with Rab5‐specific antibody and counterstained with DAPI. Scale bars, 8 μm (magnification, ×1,000). n = 5 from independent experiments. (f) The cells were pretreated with MβCD (1 mM) for 30 min before high glucose treatment (25 mM) for 48 h. Aβ 1–42 from cell culture medium were measured by human Aβ 1–42 specific ELISA assay. n = 5 from independent experiments with two technical replicates each. *P < .05, significantly different from control, # P < .05, significantly different from high glucose. Quantitative data are presented as a mean ± SEM. All blots and immunofluorescence images are representative

Article Snippet: Briefly, cells were incubated with the provided lysis buffer containing anti‐active Rab5 monoclonal antibody (NewEast Biosciences, #26911, RRID:AB_2617182).

Techniques: Western Blot, Incubation, Cell Culture, Enzyme-linked Immunosorbent Assay, Immunofluorescence

Journal: British Journal of Pharmacology

Article Title: High glucose‐mediated PICALM and mTORC1 modulate processing of amyloid precursor protein via endosomal abnormalities

doi: 10.1111/bph.15131

Figure Lengend Snippet: PICALM facilitates clathrin‐dependent APP endocytosis under high glucose conditions. (a,b) The SK‐N‐MC cells were transfected with NT siRNA or PICALM siRNA for 12 h and then treated with Pitstop 2 (30 μM) for 30 min prior to high glucose treatment (25 mM) for 24 h. (a) The cells were immunostained with APP‐ and AP2A1‐specific antibodies and counterstained with DAPI. Scale bars, 8 μm (magnification, ×1,000). n = 5 from independent experiments. (b) The cells were immunostained with APP‐ and CHC‐specific antibodies and counterstained with DAPI. Scale bars, 8 μm (magnification, ×1,000). n = 5 from independent experiments. *P < .05, significantly different from NT siRNA transfection, # P < .05, significantly different from high glucose with NT siRNA transfection. (c) Internalization assay from the cells treated with high glucose (25 mM) for 24 h was done by immunostaining with Aβ for surface‐labelled APP and PICALM specific antibodies. 0 and 15 min indicate time points of internalization. Scale bars, 8 μm (magnification, ×1,000). n = 5 from independent experiments. (d,e) The SK‐N‐MC cells were transfected with NT siRNA or PICALM siRNA for 12 h and then treated with GM6001 (20 μM) for 30 min prior to high glucose treatment (25 mM) for 24 h. (d) Cell surface was biotinylated, and labelled proteins were pulled down by streptavidin beads. Surface APP, total APP, and β‐actin were detected by western blot. n = 5 from independent experiments. (e) Biotin surface labelling and internalization assay including 15 min for internalization were done. Internalized APP, total APP, and β‐actin were detected by western blot. n = 5 from independent experiments. (f) The cells were transfected with NT siRNA or PICALM siRNA for 12 h prior to high glucose treatment (25 mM) for 48 h. Aβ 1–42 from cell culture medium were measured by human Aβ 1–42 specific ELISA assay. n = 5 from independent experiments with two technical replicates each. *P < .05, significantly different from NT siRNA transfection, # P < .05, significantly different from high glucose with NT siRNA transfection. (g) The cells were pretreated with GM6001 (20 μM) for 30 min and then treated with Dynasore (25 μM) for 30 min before high glucose treatment (25 mM) for 24 h. Cell surface was biotinylated, and labelled proteins were pulled down by streptavidin beads. Surface APP, total APP, and β‐actin were detected by western blot. n = 5 from independent experiments. (h) The cells were pretreated with Dynasore (25 μM) for 30 min immunostained with Rab5‐specific antibody and counterstained with DAPI. Scale bars, 8 μm (magnification, ×1,000). n = 5 from independent experiments. (i) The cells were incubated with Dynasore (25 μM) for 30 min prior to high glucose treatment (25 mM) for 48 h. Aβ 1–42 from cell culture medium were measured by human Aβ 1–42 specific ELISA assay. n = 5 from independent experiments with two technical replicates each. Logarithmic transformations were performed for homogeneity of the sample variance. *P < .05, significantly different from control, # P < .05, significantly different from high glucose . Quantitative data are presented as a mean ± SEM. All blots and immunofluorescence images are representative

Article Snippet: Briefly, cells were incubated with the provided lysis buffer containing anti‐active Rab5 monoclonal antibody (NewEast Biosciences, #26911, RRID:AB_2617182).

Techniques: Transfection, Immunostaining, Western Blot, Cell Culture, Enzyme-linked Immunosorbent Assay, Incubation, Immunofluorescence

Journal: British Journal of Pharmacology

Article Title: High glucose‐mediated PICALM and mTORC1 modulate processing of amyloid precursor protein via endosomal abnormalities

doi: 10.1111/bph.15131

Figure Lengend Snippet: High glucose impairs endosomal clearance via AMPK/mTORC1‐mediated autophagy defect. (a) The hippocampal samples were obtained from ZLC and ZDF rats. LC3, P62, and β‐actin were detected by western blot. n = 5 in each group. *P < .05, significantly different from ZLC rats. (b) The hippocampal samples were obtained from vehicle‐ and STZ‐treated mice. LC3, P62, and β‐actin were subjected to western blot. n = 5 in each group. *P < .05, significantly different from vehicle‐treated mice. (c) The SK‐N‐MC cells were treated with high glucose (25 mM) in a time response. p‐AMPKα (Thr172), t‐AMPKα, p‐mTOR (Ser2448), t‐mTOR, and β‐actin were detected by western blot. n = 5 from independent experiments. (d,e,g) The cells were incubated with Rapamycin (200 nM) for 30 min before high glucose treatment (25 mM) for 24 h. (d) LC3, P62, and β‐actin were subjected to western blot. n = 5 from independent experiments. (e) The cells were immunostained with LC3‐specific antibody and stained with Lysotracker red and DAPI. Scale bars, 8 μm (magnification, ×1,000). Exploratory data. (f) The cells were pretreated with PF‐4708671 (10 μM) or Rapamycin (200 nM) for 30 min prior to high glucose treatment (25 mM) for 24 h. Rab5 and β‐actin were detected by western blot. n = 5 from independent experiments. (g) The cells were immunostained with Rab5‐specific antibody and counterstained with DAPI. Scale bars, 8 μm (magnification, ×1,000). n = 5 from independent experiments. (h) The cells were pretreated with Rapamycin (200 nM) for 30 min before high glucose treatment (25 mM) for 48 h. Aβ 1–42 from cell culture medium were measured by human Aβ 1–42 specific ELISA assay. n = 5 from independent experiments with two technical replicates each. Logarithmic transformations were performed for homogeneity of the sample variance. *P < .05, significantly different from control, # P < .05, significantly different from high glucose. Quantitative data are presented as a mean ± SEM. All blots and immunofluorescence images are representative

Article Snippet: Briefly, cells were incubated with the provided lysis buffer containing anti‐active Rab5 monoclonal antibody (NewEast Biosciences, #26911, RRID:AB_2617182).

Techniques: Western Blot, Incubation, Staining, Cell Culture, Enzyme-linked Immunosorbent Assay, Immunofluorescence

Journal: British Journal of Pharmacology

Article Title: High glucose‐mediated PICALM and mTORC1 modulate processing of amyloid precursor protein via endosomal abnormalities

doi: 10.1111/bph.15131

Figure Lengend Snippet: ROS‐ and mTORC1‐mediated lysosomal dysfunction aggravates endosomal clearance and up‐regulates Aβ production. (a,j) The SK‐N‐MC cells were treated with high glucose (25 mM) for 24 h. (a) The cells were immunostained with Rab5‐specific antibody and stained with Lysotracker red and DAPI. n = 7 from independent experiments. (b) The cells treated with DW or high glucose (25 mM) were labelled with Lysotracker red and analysed by flow cytometry in a time response. n = 5 from independent experiments. *P < .05, significantly different from control. (c) The hippocampal samples were obtained from ZLC and ZDF rats. LAMP1 and β‐actin were detected by western blot. n = 5. *P < .05, significantly different from ZLC rats in each group. (d) The hippocampal samples were obtained from vehicle‐ and STZ‐treated mice. LAMP1 and β‐actin were subjected to western blot. n = 5 in each group. *P < .05, significantly different from vehicle‐treated mice. (e) The cells in a time response with 25 mM high glucose were subjected to western blot. LAMP1 and β‐actin were detected. n = 5 from independent experiments. Logarithmic transformations were performed for homogeneity of the sample variance. (f,h) The cells were pretreated with NAC (4 mM) for 30 min before high glucose treatment (25 mM) for 24 h. (f) LAMP1 and β‐actin were detected by western blot. n = 5 from independent experiments. Logarithmic transformations were performed for homogeneity of the sample variance. (g,i) The cells were incubated with Rapamycin (200 nM) for 30 min prior to high glucose treatment (25 mM) for 24 h. (g) LAMP1 and β‐actin were detected by western blot. n = 5 from independent experiments. (h,i) Intact lysosomes and intracellular pH were measured by Lysotracker red and BCECF‐AM staining, respectively. n = 5 from independent experiments. (j) The cells were immunostained with CTSB‐specific antibody and stained with Lysotracker red. (k) The cells were pretreated with leupeptin (100 nM) for 30 min before high glucose treatment (25 mM) for 48 h. Aβ 1–42 from cell culture medium were measured by human Aβ 1–42 specific ELISA assay. n = 5 from independent experiments with two technical replicates each. *P < .05, significantly different from control, # P < .05, significantly different from high glucose. Quantitative data are presented as a mean ± SEM. All blots and immunofluorescence images are representative

Article Snippet: Briefly, cells were incubated with the provided lysis buffer containing anti‐active Rab5 monoclonal antibody (NewEast Biosciences, #26911, RRID:AB_2617182).

Techniques: Staining, Flow Cytometry, Western Blot, Incubation, Cell Culture, Enzyme-linked Immunosorbent Assay, Immunofluorescence

Journal: British Journal of Pharmacology

Article Title: High glucose‐mediated PICALM and mTORC1 modulate processing of amyloid precursor protein via endosomal abnormalities

doi: 10.1111/bph.15131

Figure Lengend Snippet: Increases in early endosomes, Aβ and cognitive impairment are reversed by Dynasore and Rapamycin, respectively, in STZ‐induced DM mouse model. (a) Scheme of the experimental protocols in vivo. (b–h) The experimental mice were treated with vehicle, STZ, Dynasore, or Rapamycin, as described in Section 2. (b,e) Hippocampal slides for IHC were immunostained with Rab5‐specific antibody and counterstained with DAPI. Scale bars, 50 μm (magnification, ×200). n = 5 in each group. (c,g) Hippocampal Aβ 1–42 were measured by rat and mouse Aβ 1–42 specific ELISA assay. n = 5 in each group with two technical replicates each. (d,h) Mice were subjected to Y‐maze test to evaluate cognitive function. n = 6 in each group. (f) Hippocampal Rab5 and β‐actin were subjected to western blot. n = 5 in each group. *P < .05, significantly different from vehicle‐treated mice, # P < .05, significantly different from STZ‐treated mice. Quantitative data are presented as a mean ± SEM. All blots and immunofluorescence images are representative

Article Snippet: Briefly, cells were incubated with the provided lysis buffer containing anti‐active Rab5 monoclonal antibody (NewEast Biosciences, #26911, RRID:AB_2617182).

Techniques: In Vivo, Enzyme-linked Immunosorbent Assay, Western Blot, Immunofluorescence

Journal: PloS one

Article Title: ARHGAP22 localizes at endosomes and regulates actin cytoskeleton.

doi: 10.1371/journal.pone.0100271

Figure Lengend Snippet: Figure 4. Colocalization of ARHGAP22 with endocytic markers. (A) A7 cells were transfected with HA-ARHGAP22. After 24 h, the cells were fixed and stained with anti-HA antibody for HA-ARHGAP22 (green) and antibodies for Rab11, Rab5, EEA1, LAMP-1, GM130, or TGN46 (red). Merged fluorescent images are shown. The cells were also stained with hoechst 33258 for nuclei (blue). Scale bar, 20 mm. Insets show magnification images of the boxed regions. (B) C2C12 cells were transfected with HA-ARHGAP22. After 24 h, the cells were fixed and stained with anti-HA antibody for HA-ARHGAP22 (green) and antibodies for Rab11 or Rab5 (green). Merged fluorescent images are shown. The cells were also stained with hoechst 33258 (blue). Scale bar, 20 mm. Insets show magnification images of the boxed regions. doi:10.1371/journal.pone.0100271.g004

Article Snippet: Mouse antiLAMP-1 monoclonal and Rabbit anti-Rab5 polyclonal antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Transfection, Staining

Journal: bioRxiv

Article Title: Plasma membrane mediated GLUT10 mitochondrial targeting regulates intracellular ascorbic acid homeostasis

doi: 10.1101/2025.01.09.632083

Figure Lengend Snippet: ( A ) Quantification of the percentages of GLUT10/GFP colocalized with RAB5, RAB7 and RAB11 from Supplemental figure 8. Data are shown as mean ± SEM, n = total 6 independent experiments with 10 cells per group in each experiment. ( B ) Immunoblot to analyze the presence of RAB5 and GLUT10/V5 in GLUT10-containing vesicles. GLUT10-containing vesicles were pulled down from GLUT10/V5 expressing T-REx-293 cells with V5 antibody. ( C ) Immunoblot to analyze the presence of GLUT10/V5, RAB5 and TIM50 in mitochondria-enriched fractions. RAB5 vesicles were pulled down with RAB5A antibody from mitochondria-enriched fractions. GLUT10/V5 expression was induced in T-REx-293 cells for 12 h. ( D-F ) Oxidative stress increases GLUT10 and RAB5 in plasma membrane area. ( D ) TIRF images of plasma membrane area of GLUT10/GFP (green) and RAB5A/mCherry (magenta) in GLUT10/GFP and RAB5A/mCherry expressing MOVAS cells. ( E ) Mean fluorescence intensity of GLUT10 and RAB5 in plasma membrane area before and after treatment with 50 µM of H 2 O 2 . ( F ) Ratio of colocalization of GLUT10 with RAB5 vesicles before and after H 2 O 2 treatment. Imaging was performed at a rate of 6 frames/min (10 s/frame). Scale bar, 15 μm. ( G and H ) Oxidative stress increases GLUT10 colocalized with EEA1. ( F ) Confocal images of GLUT10/GFP colocalized with EEA1. MOVAS cells expressing GLUT10/GFP were treated with 50 µM of H 2 O 2 for 24 h and stained by immunofluorescence for EEA1. Green, GFP; magenta , EEA1; white, merged. Scale bar, 5 µm. ( G ) Quantification of GLUT10 colocalized with EEA1, as in F . Data are shown as mean ± SEM, n = total 9-10 independent experiments with 10 cells per group in each experiment. ( I and J ) Oxidative stress increases RAB5 colocalized with mitochondria. ( H ) Confocal images of RAB5 colocalized with ATP5A1 (Mito). MOVAS cells were treated with 50 µM of H 2 O 2 for 24 h and stained by immunofluorescence for RAB5 and ATP5A1. Green, RAB5; magenta , ATP5A1; white, merged. Scale bar, 5 µm. ( I ) Quantification of GLUT10 colocalized with mitochondria as in H . Data are shown as mean ± SEM, n = total 10 -12 independent experiments with 10 cells per group in each experiment. Statistical comparisons were made with two-tailed Student’s t-test in G and I , ** P < 0.01.

Article Snippet: Primary antibodies included the following: V5 (R960-25, Invitrogen), GFP (ab6556, abcam), hGLUT10 (ab110528, Abcam), TIM50 (22229-1-AP, Proteintech), TOM20 (sc-11415, Santa Cruz Biotechnology), ATP5A1/mitochondrial Complex V (459240, Invitrogen), calreticulin (06-661, Merck), KDEL (ADI-SPA-827, Enzo), beta-actin (GTX110564, GeneTex), GAPDH (10494-1-AP, Proteintech), RAB5 (ab18211, Abcam), RAB5A (11947-1-AP, Proteintech), and EEA1 (ab109110, abcam).

Techniques: Western Blot, Expressing, Membrane, Fluorescence, Imaging, Staining, Immunofluorescence, Two Tailed Test

Journal: eLife

Article Title: Increased expression of heme-binding protein 1 early in Alzheimer's disease is linked to neurotoxicity

doi: 10.7554/eLife.47498

Figure Lengend Snippet:

Article Snippet: Antibody , anti-Rab5 (Mouse, monoclonal) , Synaptic Systems , Cat#: 108 111 RRID: AB_2619777 , WB (1:1000).

Techniques: Sequencing, Isolation, Transfection, Construct, Plasmid Preparation, Retroviral, Virus, Luciferase, Negative Control, Knock-Out, Recombinant, Concentration Assay, Membrane, Software