at map over the 48 h of BW720c treatment. Loss of parasite protein production was also indicated as Western blot analysis over a 72 h BW720c time course using antibodies specific for parasite proteins, Tasp and an HSP90-like polypeptide expressed at the macroschizont stage, showed a clear reduction in levels. It can be concluded that a clear outcome of killing the parasite in the infected TBL20 lymphosarcoma line is a cessation of host cell division together with initiation of cellular processes leading to cell death. To achieve this we compared the transcriptome of the immortalised bovine cell line, BL20, with that of TBL20; followed by profiling the transcriptional changes induced by treatment of TBL20 cells with BW720c at 24 and 48 hours. BL20 cultures were also BW720c treated and analysed in parallel to exclude alterations in gene purchase Vatalanib expression caused by a direct effect of the drug on uninfected host cells. Thus, the entire experimental dataset represented differentially expressed genes from the following four pair-wise comparisons: BL20 v TBL20, TBL20 v TBL20_24 h_BW720c, TBL20 v TBL20_48 h_BW720c and TBL20_24 h_BW720c v TBL20_48 h_BW720c. RP analysis of the pair-wise comparison between BL20 and TBL20 generated a dataset of 3,079 differentially expressed genes with 1,822 elevated and 1,257 +4.9 21.7 +2.6 +3.8 +2.7 23.2 21.8 Various components of the AP1 transcription factor complex that were 23692283 elevated in TBL20 or repressed in TBL20 compared to BL20. The BW720c response is indicated to the right. The FDR generated by RP for FOSB and ATF7IP in TBL20 compared to BL20 were above our normal threshold of 5% but were included in this table as both showed a strong reversible BW720c response. doi:10.1371/journal.pone.0066833.t004 T annulata Reconfigures Host Cell Gene Expression repressed in TBL20 compared to BL20. As expected, this number of alterations was considerably higher than the previous study investigating modulation of an LPS response associated with T. annulata infected leukocytes, a large number of which showed no significant difference in expression between TBL20 and BL20. Thus, the current study represents a more comprehensive analysis of host cell gene expression changes associated with the infected leukocyte. After 48 h exposure to BW720c, RP analysis indicated that 37% of elevated genes and 39% of repressed genes in TBL20 showed statistically significant reversal. A number of previously reported bovine genes whose expression is altered at RNA or protein level by T. annulata or T. parva infection were also identified in our study and serve to validate the array analysis. Of the genes showing elevated expression in parasite-infected TBL20 cells, the most highly up-regulated encodes the chemokine XCL2, a member of a large gene family, many of which exhibit alteration associated with the Theileria-infected cell. Members of the matrix metalloproteinase family are also found to be highly up-regulated, e.g. MMP9 which was previously identified as up-regulated 
in parasite infected TBL20 and shown to be linked to metastasis of Theileria infected cells, was elevated 192 fold. Expression of MMP13, 14 and 25 were also greatly elevated in the BL20-TBL20 model. Thus our study provides a more comprehensive profile of MMP expression that is likely to contribute to the metastatic 2572306 potential of the Theileria infected cell, although variable expression of family members across different infected cell lines is likely to operate. 1,257 genes showed
