Spiroepoxide

CD1 (ICR) mice were purchased from Charles River Inc. (Wilmington, MA, USA). An N-SMase inhibitor, spiroepoxide (Santa Cruz), was intraperitoneally injected into 50-day-old male mice at 3.5 mg/kg every two day. Fourteen days later, mice treated with spiroepoxide were euthanized and the small intestine sections were subjected to small EV collection or immunofluorescence analysis as described previously [24 (link)]. All animal care was performed according to the protocols approved by the Committee for the Use and Care of Experimental Animals of the Japanese Foundation for Cancer Research (No.17-02-1, 31 5 2017).

Trans-well co-cultures were carried out in 12-well plates using Cell Culture Insert Falcon Membrane (25 mm diameter, 0.4 μm pore size, Becton Dickinson). They were set up by putting 10⁶ F12/Hut-78 or parental Hut-78 cells in the upper chamber, while 2 × 10⁶ unstimulated CD4⁺ T lymphocytes were seeded in the bottom chamber. The co-cultures were then run overnight in the presence or not of 1 μM of the inhibitors of exosome release GW4869 (Sigma) and Spiroepoxide (Santa Cruz). Then, CD4⁺ T lymphocytes were recovered and infected with HIV-1, washed, and left in culture 3 days in the presence or not of 10 μM AZT. Afterwards, lymphocytes were analyzed by FACS for the HIV-1 expression through the detection of intracytoplasmic HIV-1 CAp24.

For semiquantitative detection of EVs in vitro, anti-mouse CD63 antibodies (Y-18, Santa Cruz Biotechnology) were coupled to 4 μm aldehyde/sulfate latex beads (Invitrogen) by incubating 35 μg of v with 1 × 10⁸ beads, followed by blocking of remaining activated groups with 4% BSA in PBS44 (link).
In all, 2 × 10⁵ MC38 tumour cells were seeded per well into 24-well plates in RPMI 1640 with 10% FCS in the presence of 0, 5 and 10 μM spiroepoxide, an inhibitor of neutral sphingomyelinase 2 (Santa Cruz Biotechnology). Twenty-four hours later, these supernatants were collected. Cell culture supernatants were filtered through 0.22-μm filters. Filtrates (100 μl) were incubated with 20,000 anti-CD63-coupled beads overnight at room temperature with gentle shaking. Beads were washed and incubated with phycoerythrin-anti-CD9 (MZ3, BioLegend, San Diego, CA, USA) for 30 min on ice. After washing in 2% BSA in PBS, the beads were analysed using FACS.
For inhibition of EV production in vivo, on day 0, 2-month-old mice were intraperitoneally injected with spiroepoxide in 200 μl PBS (2 g kg⁻¹ body weight per mouse) or 200 μl of 3.75% DMSO PBS control every 48 h for 12 days (six injections total). Mice were killed 24 h after the final injection.