Fig 1: Inhibition of cholesterol and sphingomyelin affected RABV budding and release.(A) Effect of MβCD on BSR cell viability. BSR cells seeded in 96-well microplates were untreated or pre-treated with 0.5–8 mM MβCD for 1 h. The supernatants were removed and washed twice with PBS. An MTT assay was then performed. (B) Effect of MβCD on cholesterol and RABV replication. Cells seeded in six-well microplates were infected with rRC-HL at an MOI of 0.001 and then treated with 0.5–8 mM MβCD for 24 h at 37 °C. The supernatants were harvested for viral titration. The cells were assayed for cholesterol content using a cholesterol quantitation kit (40006; AAT Bioquest, Inc.) according to the manufacturer’s specfications. (C) Effect of MβCD on RABV replication. Based on A and B, a set of the cells was used to prepare cell lysates that were subjected to Western blot analysis for RABV N and β-actin protein expression. (D) The N protein/actin ratios in Fig. 3C were measured using Li-Cor Odyssey 3.0 analytical software version 29. The error bars were calculated from at least 3 independent inhibition tests. (E) Effect of myriocin on BSR cell viability. BSR cells were seeded in 96-well microplates and either untreated or pre-treated with 0.01–100 μm myriocin for 1 h. The supernatants were removed and washed twice with PBS. An MTT assay was then performed. (F) Effects of myriocin on sphingomyelin content and RABV replication. Cells seeded in six-well microplates were infected with rRC-HL at an MOI of 0.001 and then treated with 0.5–50 μm myriocin for 24 h at 37 °C. The supernatants were harvested for viral titration. The cells were assayed for sphingomyelin content using a sphingomyelin colorimetric assay kit (10009928). (G) Effect of myriocin on RABV replication. Based on D and E, another set of cells was used to prepare lysates that were then subjected to Western blotting analysis for RABV N and β-actin protein expression. (H) The N protein/actin ratios in Fig. 3G were measured using Li-Cor Odyssey 3.0 analytical software version 29.