Journal: The Journal of Biological Chemistry

Article Title: A proteolytic C-terminal fragment of Nogo-A (reticulon-4A) is released in exosomes and potently inhibits axon regeneration

doi: 10.1074/jbc.RA119.009896

Figure Lengend Snippet: The Nogo-66 loop region on the surface of exosomes. A and B, HEK293T cells were transfected with Nogo-A–Myc. 24 h after transfection, media were changed. DMSO, MG101 (20 μm), Z-VAD-fmk (20 μm), E64d (20 μm), and NH4Cl (20 mm) were added and cultured for further 12 h. Then culture supernatants were collected, and the exosome fraction was immunoblotted with anti-Myc and anti-CD9 antibodies (A). The graph shows Myc intensity divided by CD9 intensity compared to DMSO control (B). Mean ± S.E., n = 5–8 independent experiments. *, p < 0.05; ***, p < 0.005; one-way ANOVA followed by Dunnett's test. C and D, HEK293T cells were transfected with Nogo-A–Myc. 24 h after transfection, media were changed. DMSO, the indicated amounts of BACE inhibitors, and NH4Cl (20 mm) were added and cultured for further 12 h. Then culture supernatants were collected, and the exosome fraction was immunoblotted with anti-Myc and anti-CD9 antibodies (C). The graph shows Myc intensity divided by CD9 intensity compared to DMSO control (D). Mean ± S.E., n = 4 independent experiments. **, p < 0.01; ***, p < 0.005; one-way ANOVA followed by Dunnett's test. E, HEK293T cells were transfected with Nogo-A–Myc and siNC, siBACE1 #1, or siBACE1 #2. Exosomes were purified 36 h after transfection and immunoblotted with anti-Myc and anti-CD9 antibodies. F, quantification of Myc intensity divided by CD9 intensity compared to DMSO control. Mean ± S.E., n = 6 independent experiments. **, p < 0.01; ***, p < 0.005; one-way ANOVA followed by Dunnett's test. G, real-time PCR for replicates of siBACE-transfected HEK293T cells. BACE1 mRNA expression was normalized to Gapdh mRNA expression. Mean ± S.E., n = 4 independent experiments. ***, p < 0.005; one-way ANOVA followed by Dunnett's test.

Article Snippet: MG101 (R&D Systems), Z-VAD-FMK (Promega), E64d (Santa Cruz Biotechnology), and β-secretase inhibitor IV (Merck) were used for inhibitor experiments.

Techniques: Transfection, Cell Culture, Control, Purification, Real-time Polymerase Chain Reaction, Expressing

Journal: bioRxiv

Article Title: OrgaMeas: A pipeline that integrates all the processes of organelle image analysis

doi: 10.1101/2024.04.15.589634

Figure Lengend Snippet: (A) Representative images of HeLa cells deprived of amino acids and FBS (ST: starvation) in the presence or absence of protease inhibitors (PI), 10 μg/mL pepstatin A, and 10 μg/mL E-64d, for 1 h. Total lysosomes and activated lysosomes were stained with the pH-insensitive probe LysoPrime Green (left six panels) and the pH-sensitive probe LysoTracker Red (right six panels), respectively. Scale bar, 20 µm. Lower panels are magnified images of the regions surrounded by yellow dotted lines in the respective upper panels. Representative data from three independent experiments are shown. (B, C) Quantification of the lysosomal area and average diameter of individual lysosomes per cell (B) and the intensity of LysoTracker Red signal per cell (C) using OrgaMeas. More than 100 cells were analyzed per experimental group. Data are presented as individual means. ***p < 0.001, Dunnett’s multiple comparison test (Ct vs ST or ST + PI) in (B), Tukey’s multiple comparison test (Ct vs ST, Ct vs ST + PI, ST vs ST + PI) in (C). Representative data from three independent experiments are shown.

Article Snippet: CCCP, pepstatin A, and E-64d were purchased from Fujifilm Wako Chemicals (Osaka, Japan).

Techniques: Staining, Comparison