Journal: PLoS pathogens

Article Title: A cytoplasmic tail determinant in HIV-1 Vpu mediates targeting of tetherin for endosomal degradation and counteracts interferon-induced restriction.

doi: 10.1371/journal.ppat.1002609

Figure Lengend Snippet: Figure 4. Vpu ELV interacts and colocalizes with tetherin, and is incorporated into nascent virions. (A) 293T/tetherin cells were transfected twice over 48 h with siRNA oligonucleotide directed against UBAP1 or Non-targeting control. The cells were then infected with the indicated virus at an MOI of 2. 48 h later cell lysates were immunoprecipitated with an anti-tetherin monoclonal antibody. Lysates and immunoprecipitates were separated by SDS-PAGE and blotted for tetherin, UBAP1 or Vpu using LiCor quantitative Western blotting. Numbers under the Vpu lanes of the cell lysate represent relative band intensities. The histogram below the IP represents the ratio of tetherin band intensity to that of Vpu in the co-IP. (B) HeLa and 293T/tetherin cells were infected as in Figure 3 and stained for Vpu (green) and tetherin (red) and examined by confocal microscopy. Adjacent histograms quantify the degree of co-localization of Vpu ELV and tetherin (Pearson’s Correlation Coefficient calculated using ImageJ) for 20 individual cells. (C) and (D) 293T or 293T cells stably expressing tetherin delGPI were infected with HIV-1 wt, HIV-1 Vpu ELV or HIV-1 delVpu at an MOI of 1. 48 h post infection, cells were harvested and viral supernatants were pelleted through a 20% sucrose cushion. Cells and virions were subjected to SDS-PAGE and Western blotting for tetherin delGPI, Vpu, HIV-1 p24CA and Hsp90 and analyzed by LiCor quantitative imager. doi:10.1371/journal.ppat.1002609.g004

Article Snippet: Virion and cell lysates were then subjected to SDS-PAGE and Western blotted for HIV-1 p24CA (monoclonal antibody 183-H12-5C; kindly provided by B Chesebro through the NIH ARRP), rabbit anti-Hsp90 (Santa Cruz Biotechnologies), monoclonal mouse anti-HA.11 (Covance), polyclonal rabbit anti-HA (Rockland) and/or Vpu (rabbit polyclonal; kindly provided by K. Strebel through the NIH ARRP [67], and visualized by LiCor apparatus using fluorophores conjugated secondary antibodies (IRDye 800 Goat anti-rabbit, IRDye 680 Goat anti-mouse).

Techniques: Transfection, Control, Infection, Virus, Immunoprecipitation, SDS Page, Western Blot, Co-Immunoprecipitation Assay, Staining, Confocal Microscopy, Stable Transfection, Expressing

Journal: Advanced Science

Article Title: Melanoma Derived Exosomes Amplify Radiotherapy Induced Abscopal Effect via IRF7/I‐IFN Axis in Macrophages

doi: 10.1002/advs.202304991

Figure Lengend Snippet: CircPIK3R3/IRF7/I‐IFN axis participates in the combination of radiotherapy and anti‐PD1 mediated abscopal effect. A) The treatment model involving RT, anti‐PD1, and RO8191: C57/BL6 mice were subcutaneously inoculated with 1 × 106 sh‐NC B16F10 cells or sh‐circ‐0011074 B16F10 cells. On day 5, C57/BL6 mice were intravenously injected with 1 × 106 B16F10‐luc cells. Starting from day 6, mice were administered the IFN receptor agonist RO8191 via daily intraperitoneal injections at a dose of 1 mg kg −1 . On day 7, radiotherapy was initiated, with a daily dose of 8 Gy administered for 3 consecutive days. On day 7, mice were also administered anti‐PD1 via intraperitoneal injection every 2 days at a dose of 100 µg/mouse until the endpoint of observation. B,C) Measurement of subcutaneous tumor weight in each group to assess treatment efficacy ( n = 3). D,E) Evaluation of fluorescent intensity in lung metastatic foci using bioluminescence imaging to assess treatment efficacy ( n = 6). F,G) Immunohistochemical examination of CD8 + T cell infiltration in subcutaneous tumors and lung metastatic foci ( n = 3). H,I) Immunofluorescence detection of IRF7 + macrophage infiltration in subcutaneous tumors and lung metastatic foci (n = 3). Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 by two‐tailed unpaired Student t‐test.

Article Snippet: Anti‐mouse PD‐1 mAb (Bio X Cell, USA) was administered intraperitoneally (200 mg per mouse) every 2 days during treatment.

Techniques: Injection, Imaging, Immunohistochemical staining, Immunofluorescence, Two Tailed Test