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: Journal of Virology

Article Title: Bovine coronavirus enters HRT-18 cells via membrane fusion and clathrin-mediated endocytosis in a low pH-, dynamin-, cholesterol-, microtubule-, Rab7-, and Rab11-dependent manner

doi: 10.1128/jvi.01274-25

Figure Lengend Snippet: BCoV entry into HRT-18 cells depends on cathepsin. ( A ) The maximum safe concentrations of E64d were determined using the CCK-8 assay. ( B ) Western blot analysis was used to evaluate the BCoV N protein expression levels, with grayscale analysis performed and presented as a bar graph. ( C ) RT-qPCR was performed to assess the BCoV gene copy numbers. ( D ) TCID 50 assay was used to measure the BCoV viral titers in the cell supernatant. ( E ) IFA was used to detect the number of BCoV-infected cells. Scale bar = 100 µm. ( F ) RT-qPCR was used to evaluate the effect of E64d on the viral entry. ( G ) RT-qPCR was used to evaluate the effect of E64d on BCoV attachment. Data are presented as the mean ± SD of three independent experiments (not significant, P > 0.05; * P < 0.05; ** P < 0.01; *** P < 0.001).

Article Snippet: The inhibitors used in this study included SSAA09E3 (Cat HY-138102, MedChemExpress), a novel inhibitor that blocks the fusion of the viral membrane with the host cell membrane; CPZ (Cat C0982, Sigma), a clathrin-mediated endocytosis inhibitor; nystatin (Cat 475914, Sigma), a caveolae inhibitor that acts as a sterol-binding agent disrupting caveolae; blebbistatin (Cat 203391, Sigma), an inhibitor of micropinocytosis; dynasore (Cat T1848, TargetMol), a dynamin inhibitor; MβCD (Cat T4072, TargetMol), a cholesterol depletion inhibitor; chloroquine (Cat S6999, Selleck) and NH 4 Cl (Cat A9434, Sigma), a potent inhibitor of V-ATPase and a specific inhibitor of acidification of endosomal vesicles; colchicine (Cat HY-16569, MedChemExpress), which inhibits the polymerization of tubulin; E64d (Cat S7393, Selleck), a cathepsin inhibitor; and camostat (Cat HY-13512, MedChemExpress), a TMPRSS2 inhibitor.

Techniques: CCK-8 Assay, Western Blot, Expressing, Quantitative RT-PCR, Infection

Journal: The FEBS journal

Article Title: Verapamil treatment induces cytoprotective autophagy by modulating cellular metabolism.

doi: 10.1111/febs.14064

Figure Lengend Snippet: Fig. 7: Verapamil induces autophagic flux Western blots for LC3 protein levels after treatment with increasing concentrations of Verapamil (Ver; 50, 100, 200 µM; 6 h) alone or in combination with lysosomal protease inhibitors – E64d/Pepstatin A (E/PepA; 10 µM; 6 h) (A-E). Actin or ERK2 were used as a loading control. Representative images of the Western blots are shown (upper panels) together with the quantification of LC3-II fold increase in comparison to control, untreated cells (lower panels). Each performed experiment is presented as an individual data point set with mean (±SEM), n= 3 to 6.

Article Snippet: Verapamil chloride (Sigma-Aldrich, V4629), E64d (LKT Laboratories, E0003), Pepstatin A (Calbiochem, 516481), Chloroquine (Sigma-Aldrich, C6620), Sodium oxamate (Sigma-Aldrich, O2751).

Techniques: Western Blot, Control, Comparison