Figure 2  Inhibition of BSRV replication by cyclopamine in cell culture.

A Amino acid sequence alignment of M2-1 of HRSV strain Long and BRSV Lövsta strain (GenBank accession codes AMA66581.1 and QBK50987.1, respectively) by ClustalW and prepared with ESPript3. The R151 residue is indicated by an arrow. RNA and P-binding domains previously identified [20, 23], are underlined by red and blue lines, respectively. B BT cells in 96-well plates were infected with rBRSV-mCherry at MOI 0.02 and incubated in the presence of serial dilutions of CPM (initial concentration of 10 µM, serial dilutions at ½). The mCherry fluorescence was quantified on live cells at 7 dpi using a Tecan infinite M200Pro spectrofluorometer. Data were normalized by the value of fluorescence of untreated infected cells. Data are representative of three experiments made in duplicates. Standard deviations are indicated. C Images of BT cells infected with rBRSV-mCherry and treated with CPM at different concentrations at 7 dpi. Cell nuclei were stained with Hoechst 33342. Scale bar, 100 μm. D BT cells in 96-well plates were infected with either Lövsta or rBRSV-mCherry at MOI 0.02 and incubated in the presence of serial dilutions of CPM (initial concentration of 10 µM, serial dilutions at ½). Cells were fixed at 7 dpi, immunolabelled with anti-F antibody, followed by incubation with secondary antibody coupled to Alexa488. Fluorescence intensity was measured using a Tecan infinite M200Pro spectrofluorometer. Data were normalized by the value of fluorescence of corresponding untreated infected cells. Data are representative of two experiments made in duplicates. Standard deviations are indicated.