T 705

Bryostatin analogs (1–3) were synthesized as described before [11 (link),12 (link),14 (link)]. T-705 was obtained from Boc Sciences (New York, USA). Go 6976 was purchased from Abcam Biochemicals (Cambridge, UK) and sotrastaurin from Axon Medchem BV (The Netherlands). All compounds were dissolved in analytical grade DMSO to yield 10 mg/ml stocks. The compounds were protected from light and were stored at −20°C until used. Arbidol was a kind gift by Prof. S. Polyak (University of Washington, Seattle, USA) and was dissolved as a 10 mg/ml solution in 100% ethanol after which it was 10-fold diluted with sterile water before use.

All compounds were obtained from either Selleckchem or Sigma-Aldrich with the exception of T-705 (BOC Sciences). Cells were seeded at either 1 × 10⁴ cells/well (96-well plate) or 4 × 10³ cells/well (384-well plate) 16–20 h prior to treatment with DMSO-diluted compounds (final DMSO concentration 0.5%). For the 384-well assay, compounds were added to the plate using an Echo acoustic liquid handler (Labcyte). Cells were infected 1 h post treatment, and at 72 hpi, either supernatants were collected or ZsG fluorescence was determined using a BioTek Synergy reader (height 6 mm; 100 gain/sensitivity). Cell viability (ATP content) was determined in parallel on compound-treated, mock-infected cells using CellTiter-Glo 2.0 (Promega). All experiments were performed in quadruplicate and repeated at least 3 times.