Figure 2
From: ROCK1/MLC2 inhibition induces decay of viral mRNA in BPXV infected cells
Effect of Thiazovivin on levels of BPXV mRNA/protein/DNA. (a) Viral mRNA. Confluent monolayers of Vero cells, in triplicates, were infected with BPXV (MOI of 5), followed by washing with PBS and addition of fresh medium. Inhibitor or vehicle-controls were applied at 4 hpi. Cells were scraped at 12 hpi to isolate RNA. cDNA was synthesized using oligo (dT) primers and quantified for BPXV M gene by qRT-PCR. Ct values were normalized with the β-actin housekeeping control gene and relative fold change was calculated by the ∆∆ Ct method. n = 3 independent experiments. (b) Viral proteins. Cells were infected with BPXV and treated with Thiazovivin as described above. The cell lysates were prepared at 24 hpi. The levels of viral (upper panel) and β-Actin house-keeping control protein (lower panel) were determined by Western blot analysis (bi). The histogram (bii) shows the band intensity of the protein. The blots were quantified by densitometry (ImageJ) and the data are presented as mean with SD. n = 3 independent experiments. See Supplementary Fig. 6a for full blots. (c) Viral DNA. Vero cells, in triplicates, were infected with BPXV and treated with Thiazovivin as described above. BPXV M gene in Thiazovivin-treated and vehicle-control-treated Vero cells were quantified by qRT-PCR. Ct values were normalized with the β-actin housekeeping control gene and relative fold change was calculated by the ∆∆ Ct method. n = 3 independent experiments. Error bars indicate SD. Pair-wise statistical comparisons were performed using Student's t-test (***P < 0.001; **P < 0.01).
