Effects of C003 and C052 on BTV-induced caspase-3/7 activation.

<p>BSR cells were mock infected or infected with BTV at MOI of 0.01, and treated with C003 or C052 at their EC₉₀ concentrations (A) or EC₅₀ concentrations (B). At 24, 48 and 72 h.p.i, caspase-3/-7 activities were determined using the Caspase-Glo-3/7 reagent, and presented as relative fluorescent unit (RLU). Each data points represented mean values and SD from triplicates experiments.</p

The effects of C003 and C052 on BTV viral genomic RNA synthesized and progeny virus production at MOI of 0.01.

<p>BTV infected cells were treated with C003 (10 µM) or C052 (2.5 µM), at different h.p.i. as indicated in figures. The viral genomic RNA was extracted and the copy number was determined using real-time qRT-PCR in cell (A) and supernatant (B) samples. The progeny virus titer in cells (C) and supernatants (D) were also analyzed using standard plaque assay.</p

The compound structure of C003 was showed.

<p>The compound was divided into five zones as indicated.</p

The virostatic efficacy and cytotoxicity of C052 were showed as cells were infected with BTV at MOI of 0.01 in the presence of various concentrations of C052.

<p>At 72 h.p.i, cell viability (triangles) or cytotoxicity (circles) was determined using CellTiter-Glo reagent. Each data point represents means and SD from five replicates.</p

Effects of C003 and C052 on BTV VP6 protein expression and LC3-I/LC3-II conversion were analyzed in sample collected at different h.p.i.

<p>Cells were infected with BTV at MOI of 0.01, and treated with C003 (10 µM) or C052 (2.5 µM). VP6 expressions and LC3-I/LC3-II conversion in these samples were determined using the western blot.</p

The time-of-addition assay for C003 (A) and C052 (B).

<p>C003 at 20 and 1.76 µM, and C052 at 2.5 µM and 0.27 µM, respectively, were added to BTV infected cells at different h.p.i. as indicated, and the protection of C003 and C052 against BTV induced CPE, or cell viability, was measured using CellTiter-Glo reagent at 72 h.p.i. Each data points represented the average values and SD from of eight independent replicates.</p

The <i>de novo</i> synthesis of C052 (2a) and C055 (3a) were illustrated.

<p>Each number here represents each intermediate compound (ic) as described in the text.</p

The effects of C003 and C052 on BTV progeny production.

<p>BTV infected cells were treated with C003 (10 µM) or C052 (2.5 µM). At different time post infection as indicated in figures, cell (A) and supernatant (B) samples were collected separately. BTV progeny productions were evaluated using the standard plaque assay. In a separate experiment, the plaque reduction assay (C) was carried by mixing C003 or C052 with the overlay and applied directly to BTV-infected cells. At 72 h.p.i, plaques were counted and analyzed. Each data points represented the average values and SD from of three independent replicates.</p

Both C003 and C052 protects BSR cells from BTV induced CPE/apoptosis.

<p>In mock infected BSR cells (A), cells formed a monolayer with no cells detached from the plates. At 72 h.p.i., BTV infected cells showed CPE/apoptosis and were detached from the plate (B). When C003 at 10 µM was added to the BSR cells without BTV infection, there were no obvious changes in term of cell morphology (C). There were also no morphology changes when cells were treated with C003 and with BTV infection (D). Similarly, when C052 at 2.5 µM was added to the BSR cells without BTV infection, there were no cytotoxicity (E), and C052 could also block BTV-induced CPE/apoptosis (F).</p

The virostatic efficacy and cytotoxicity were showed in (A) C003 and (B) C001.

<p>Cells were infected with BTV at MOI of 0.01 with or without C003 or C001 at various concentrations. At 72 h.p.i, cell viability (triangles) or cytotoxicity (circles) was measured using the CellTiter-Glo reagent. Each data point represents means and standard deviation (SD) from five replicates.</p