Fig 2: Characterization of the TaPin1 antibody and secretion of TpPin1 in Theileria parvaa The antibody raised against TaPin1 specifically recognizes Theileria version in TaPin1-stably transfected NIH3T3 cells but not the empty vector in Control-stably transfected NIH3T3 cells or the human version in hPin1-stably transfected NIH3T3 cells. Both Theileria and Human versions of Pin1 are present respectively in TaPin1-stably transfected NIH3T3 and hPin1-stably transfected NIH3T3 cells.b. The antibody raised against TaPin1 specifically recognizes GST-TaPin1 but not GST and GST-hPin1 beads.c. TpPin1 protein was detected in the nuclear and cytoplasmic fractions of T.parva-infected TpMD409 cells and decreased upon Buparvaquone (Bup) treatment. “Con” indicates Control. Antibodies recognizing apicomplexan actin, bovine Tubulin or bovine Histone H3 were used as controls.d. TaPin1 expression in BL3 or parasite-infected TBL3 cells was examined by confocal microscopy analysis using an antibody raised specifically against Theileria TaPin1, counterstaining with DAPI. 3D reconstruction in BL3 and TBL3 cells are shown.e. Immunostaining of TaPin1 and HA in NIH-3T3 stably expressing TaPin1 and NIH-3T3 stably expressing hPin1 counterstaining with DAPI.All the results are representatives of 3 independent experiments.

Fig 3: Bovine Pin1 is not regulated by the parasiteBuparvaquone treatment had no effect on the mRNA levels (a) of bovine Pin1 in infected (TBL3) or non-infected (BL3) cells as assessed by qPCR analysis and on the protein level (b) as assessed by immunoblot analysis. “Con” indicates Control and “Bup” indicates Buparvaquone treatment. (average ± sd, n=3). The result in 3b is representative of 3 independent experiments.

Fig 4: Theileria parasites secrete a conserved Pin1 PPIase proteina. Expression of Pin1 RNA in T. annulata-infected TBL3 cells, uninfected BL3 cells or T. parva-infected TpMD409 cells, treated with Buparvaquone (Bup) or control (Con). Bovine Pin1 expression was used as loading control.b. TaPin1 protein was detected in the host cytoplasm and nucleus, in contrast Apicomplexan actin (TaActin). Bovine Histone H3 (nuclear) and Tubulin (cytoplasmic) proteins were controls. Relative quantification showing TaPin1/Tubulin or TaPin1/Histone H3 ratios calculated with Image J software (average ± sd, n=3). The p-values were corrected for the multiple comparisons using the Bonferroni correction based on the total overall number of pairwise comparisons. *p<0.05, **p<0.01.c. TaPin1 was detected in the cytoplasm and nucleus of infected cells by confocal microscopy using an affinity-purified antibody specific for TaPin1, counterstaining with DAPI (white arrows indicate parasites).Results are representative of 3 independent experiments.

Fig 5: Inhibition of bovine Pin1 does not affect Theileria-associated cell transformationa. Buparvaquone (Bup), Juglone (Jug) and DTM decreased the viability of cells infected with T. parva (TpMD409). Host cell viability was assessed using the XTT assay after 72h.b. Colony formation of parasite-infected TpMD409 cells in soft-agar was lost after 72h treatment with Buparvaquone (Bup), Juglone (Jug) or DTM. Number of macroscopic colonies per plate were counted after 10 days.c. siPin1 has no effect on TBL3 transformed phenotypes. TBL3 were transiently transfected with siControl or siPin1. The average number of colonies per plate is shown. (average ± sd, n=3). Indicated protein levels were detected by Western blot analysis using specific antibodies. Actin was used as loading control (Results representative of 3 independent experiments).d/f. Overexpression of the TaPin1 Mutant A53P rescued the Buparvaquone but not Juglone effects on Theileria infected cells.All data represent 3 independent experiments (average ± sd, n=3). The SPSS 19.0 program (SPSS Inc. Chicago, IL, USA) was used for statistics. *p<0.05, **p<0.01.