fab36551r  (R&D Systems)

Journal: Scientific Reports

Article Title: Cannabinoid receptor type 2 agonist JWH-133 decreases cathepsin B secretion and neurotoxicity from HIV-infected macrophages

doi: 10.1038/s41598-021-03896-3

Figure Lengend Snippet: HIV-infected MDM keep expressing surface CB2R at day 12pi. MDM were labeled with a fluorescent CB2R monoclonal antibody (FL4 channel) conjugated with Alexa Fluor® 647 and then analyzed by flow cytometry. ( a ) Representative histograms of extracellular staining show significant differences in fluorescence intensities from HIV + (dark histogram) and uninfected (grey histogram) on the expression of CB2R compared with the nonfluorescent MDM (unshaded histograms to the left. ( b ) A graphic representation of surface CB2R levels per donor is shown. Panels a and b are representative of three different donors (n = 3). An unstained control was included as a technical negative control and is representative of 1 donor (n = 1). ( c ) Representative images of intracellular levels of CB2R and GAPDH expression using Western Blot. Images were cropped from the same blot after stripping and reprobing with a different antibody and were acquired using different exposure times. Full-length blots are presented in Supplementary Fig. 4 ( d ) A graphic representation of intracellular CB2R levels per donor is shown. Figures c and d are representative of four different donors (n = 4).

Article Snippet: MDM were incubated with an anti-human CB2R-Alexa Fluor® 647 conjugated antibody (R&D Systems) (1:100 in PBS 1X) for 1 h at 4 °C.

Techniques: Infection, Expressing, Labeling, Flow Cytometry, Staining, Fluorescence, Control, Negative Control, Western Blot, Stripping Membranes

Journal: Scientific Reports

Article Title: Cannabinoid receptor type 2 agonist JWH-133 decreases cathepsin B secretion and neurotoxicity from HIV-infected macrophages

doi: 10.1038/s41598-021-03896-3

Figure Lengend Snippet: CB2R agonists treatment decrease HIV-1 replication and CATB secretion from MDM. MDM were infected with HIV-1 and treated with CB2R agonists, JWH-133 and HU-308. HIV-1 p24 and CATB levels were measured from supernatants of days 3, 6, 9, and 12pi by ELISA. ( a ) Temporal HIV-1 p24 levels in supernatants of HIV-infected MDM treated with JWH-133. ( b ) Temporal CATB secretion levels in supernatants of HIV-infected MDM treated with JWH-133. Figures a and b are representative of at least four different donors (n = at least 4). ( c ) Temporal HIV-1 p24 levels in supernatants of HIV-infected MDM treated with HU-308. ( d ) Temporal CATB secretion levels in supernatants of HIV-infected MDM treated with HU-308. Figures c and d are representative of at least six different donors (n = at least 6). Graphs are presented using the mean and the ± standard error of the mean (SEM). * p < 0.05, ** p < 0.01 vs. vehicle control at its respective time-point.

Article Snippet: MDM were incubated with an anti-human CB2R-Alexa Fluor® 647 conjugated antibody (R&D Systems) (1:100 in PBS 1X) for 1 h at 4 °C.

Techniques: Infection, Enzyme-linked Immunosorbent Assay, Control

Journal: Scientific Reports

Article Title: Cannabinoid receptor type 2 agonist JWH-133 decreases cathepsin B secretion and neurotoxicity from HIV-infected macrophages

doi: 10.1038/s41598-021-03896-3

Figure Lengend Snippet: JWH-133 prevents HIV-induced increase in surface CB2R expression and induces oscillating expressions over time. MDM were cultured in 8-well chamber slides, infected with HIV-1 ADA , and treated with JWH-133 at 0.5 µM. Slides were fixed at days 3, 6, 9, and 12dpi. Cells were stained with an anti-CB2R antibody (red) and nuclei were stained with DAPI (blue). At least 3 different random pictures per condition were acquired. Representative immunofluorescence images are shown. Graphs are presented using the mean ± standard error of the mean (SEM). This figure is representative of three different donors (n = 3). * p < 0.05, ** p < 0.01.

Article Snippet: MDM were incubated with an anti-human CB2R-Alexa Fluor® 647 conjugated antibody (R&D Systems) (1:100 in PBS 1X) for 1 h at 4 °C.

Techniques: Expressing, Cell Culture, Infection, Staining, Immunofluorescence

Journal: Scientific Reports

Article Title: Cannabinoid receptor type 2 agonist JWH-133 decreases cathepsin B secretion and neurotoxicity from HIV-infected macrophages

doi: 10.1038/s41598-021-03896-3

Figure Lengend Snippet: JWH-133 decreases HIV-1 replication and CATB secretion through CB2R activation. MDM were cultured in 24-wells plates and infected with HIV-1 ADA . After removal of residual virus, cells were treated with CB2R antagonist SR144528 (SR: 1 µM) for 1 h, followed by JWH-133 (JWH) treatment at 0.5 µM. Treatments were maintained for 6dpi, exchanging half of the media at day 3pi and repeating the co-administration protocol. HIV-1 p24 and CATB levels were measured in HIV-infected MDM supernatants using ELISA. HIV-1 p24 and CATB levels were normalized against its vehicle control per donor. ( a ) HIV-1 p24 levels in HIV-infected MDM after co-administration of CB2R ligands. This figure is representative of at least eight different donors (n = 8). ( b ) CATB levels in HIV-infected MDM after co-administration of CB2R ligands. This figure is representative of ten different donors (n = 10). Graphs are presented using the mean ± standard error of the mean (SEM). * p < 0.05, ** p < 0.01.

Article Snippet: MDM were incubated with an anti-human CB2R-Alexa Fluor® 647 conjugated antibody (R&D Systems) (1:100 in PBS 1X) for 1 h at 4 °C.

Techniques: Activation Assay, Cell Culture, Infection, Virus, Enzyme-linked Immunosorbent Assay, Control