Kpt 335

RVH-421 (ACC 127) was obtained from DSMZ-German Collection of Microorganisms and Cell Cultures, and SK-MEL-5 (HTB-70) was obtained from ATCC as part of the NCI-60 cancer cell line panel. Each cell line was cultured in RPMI 1640 medium (Corning, 10–040-CV) supplemented with 10% FBS and 1% penicillin and streptomycin. Cancer cells were seeded at a concentration of 3 × 10⁵ cells into 12-well plates and incubated at 37°C overnight. Cells were washed with warm Dulbecco's PBS before being treated with 10 μM KPT-335 (Selleckchem, S7707) or 5 ng/ml leptomycin B (LMB; Sigma, L2913) in the presence or absence of 50 ng/ml IFN-γ (PeproTech, 300–02) in RPMI 1640 medium supplemented with 10% FBS and 1% penicillin and streptomycin for 32 h.

Cells were spinoculated with HIV-1 (1 h at room temperature [RT] and 1,100 × g) at various multiplicities of infection (MOI, typically 0.5 to 2), cultured for 2 to 3 h at 37°C, washed to remove unbound virus particles, and cultured for 3 to 6 days. Infection was quantified by analyzing p24^Gag released into the culture supernatants or GFP expression by flow cytometry (BD LSRII). In some experiments, cells were pretreated prior (at least 30 min) to infection with efavirenz (1 µM; NIH AIDS Reagent Program), raltegravir (30 µM; Selleck Chemicals), or treated 2 to 3 h postinfection (p.i.) with KPT-330 (1 µM, selinexor; Selleck Chemicals), or KPT-335 (0.1 µM, verdinexor; Selleck Chemicals). DMSO (Sigma-Aldrich) was used as a vehicle control.