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Fig. 7 | Retrovirology

Fig. 7

From: Latent HIV-1 is activated by exosomes from cells infected with either replication-competent or defective HIV-1

Fig. 7

Latent HIV-1 is activated by exosomes from cells infected with defective HIV-1. a A total of 120 μU of exosomes purified from the supernatants of either parental Hut-78 or Hut-78/F12 cells were used to challenge 5 × 104 U1 cells in U-bottom 96 well plates. After 24 h, cells were extensively washed, and released HIV-1 was measured as CAp24 concentration in cell supernatants after additional 24 h. As a control, cells were either left untreated (Ctrl) or treated with 100 ng/mL of recombinant TNFα. The results are expressed as mean values + SD from six independent experiments carried out with duplicates. *p < 0.05. b The same experiment whose data are reported on panel a has been reproduced on U1 treated with either TAPI-2, anti-TNFα Abs, or isotype matched IgGs soon after the exosome challenge. Shown are the results calculated as mean values from two experiments with duplicates. c Western blot analysis for the expression of ADAM17 in either Hut-78 or Hut-78/F12 cells. Signals from cellular ADAM17 were normalized with β-actin signals, whereas exosome preparations were also probed for the presence of ICAM-1. On the left of each panel, molecular weight markers are given in kDa. On the right, arrows identify both inactive and active ADAM17 forms. The results are representative of two independent experiments. d Effects of exosomes from Hut-78/F12 cells on HIV-1 latently infecting primary CD4+ T lymphocytes. A total of 5 × 104 of untouched primary CD4+ T lymphocytes was challenged by spinoculation with (VSV-G) ΔenvHIV-1 in the presence or not of AZT. As a control, conditions with unchallenged CD4+ T lymphocytes (Ctrl) were included. After 48 h, HIV-1 latently infected cells were extensively washed and then left in culture for additional 24 h. Afterwards, cells were challenged with 120 μU of exosomes from either Hut-78 or Hut-78/F12 cells. Shown are the percentages of HIV-1 Gag expressing cells as detected by intracytoplasmic CAp24 FACS analysis 24 h post-exosome challenge. As a control, cells were either treated with PMA + ionomycin (PMA), or left untreated (Ctrl). In addition, CD4+ T lymphocytes challenged with (VSV-G) ΔenvHIV-1 in the presence of AZT were treated with either PMA + ionomycin or exosomes from either Hut-78 or Hut-78/F12 cells. Shown are the results calculated as mean percentage values + SD of triplicate cultures of CD4+ T lymphocytes from three donors. *p < 0.05. e The same experiment whose data are reported in panel d has been reproduced using CD4+ T lymphocytes treated with either TAPI-2, anti-TNFα Abs, or isotype matched IgGs soon after exosome challenge. The results are shown as mean percentage values from two experiments with duplicate cultures

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