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	Figure 1. Bioinformatic analysis. A. Comparison between the deduced amino acid sequence of P. falciparum inhibitor 2 (PfI2) and Human inhibitor 2. (PlasmoDB gene identifier: PF3D7_0320000) was aligned with the Human Inhibitor 2 (HuI2 (Genbank accession number: NM_006241) homologous using the ClustalW algorithm and was manually corrected. The identical residues are shown by a star. Amino acids are numbered at the right of the sequence. The first box contains the “KGILK” motif, the second the “RvXF” motif and the third, the “HYNE” motif. The amino acids involved in the putative nuclear localization signal are shown in light gray. B. Known interaction sequences between Human I2 and PP1 were compared to PfI2 sequences. C. Predicted PfI2 structure. The helices are represented by a cylinder, the coils are represented by a line. D. Phylogenic tree of the Inhibitor 2 family. A maximum likehood tree was generated from the 21 inhibitor-2 sequences using MEGA5 under the JTT + G + I model with 100 bootstrap repetitions. Outgroups are formed by inhibitor 3 orthologs (I3, outgroup 1) and sds22 like proteins (LRR1, outgroup 2).
	Figure 2. Expression and localization of the PfI2 gene product by P. falciparum. A. Purified His6-PfI2 separated by 15% SDS-PAGE and blotted onto nitrocellulose (Red Ponceau staining, lane 1) and revealed with mAb anti-His (lane 2) showed a single band at ~ 20 kDa, indicating an anomalous electrophoretic migration of PfI2 (expected size 16.7 kDa). The identity of the purified recombinant PfI2 was further confirmed by MALDI-TOF mass spectrometry. B. Immunoprecipitation of native PfI2 with anti-PfI2 polyclonal antibodies from Plasmodium falciparum extracts, followed by an immunoblot analysis with preimmune serum (lane 1) or with anti-PfI2 antisera (lane 2). C. Detection of PfI2 in total proteins extracted from asynchronous cultures of P. falciparum using PfPP1 column. Total protein extracts (10 mg) pre-cleared on Ni-NTA sepharose beads were incubated overnight with His6-PfPP1 affinity Ni-NTA column as described in Methods. The blots were probed with preimmune serum (lane 1), anti-PfI2 (lane 2) or with anti-His mAb antibodies (lane 3). The blots were revealed as described in Methods. D. Immunoblot analysis of pARL2-PfI2-GFP transfected P. falciparum. Protein extracted from wild-type parasites (lane 1) or from transfected parasites (lane 2) were subjected to western-blotting and probed with anti-GFP mAb antibodies. E. Expression and localization of PfI2-GFP throughout the erythrocytic cell cycle of P. falciparum. Parasites were transfected with pARL2-PfI2-GFP construct as described in Methods and live transfectants were analysed by fluorescence microscopy.
	Figure 3. Targeted gene disruption and HA-tagging of the PfI2 locus. A. Disruption of PfI2 by knock-out strategy using single homologous recombination. The pCAM-BSD construct, the blasticidin-resistance cassette (BSD), the location of the primers (Additional file 1: Table S1) used for PCR analysis and the locus resulting from integration are indicated. B. Insertion of an HA epitope tag at the PfI2 C terminus (Knock-in strategy). C. Analysis of pCAM-BSD-PfI2 transfected 3D7 culture by PCR (Knock-out strategy). Lanes 1–4 correspond to DNA extracted from wild type parasites, lanes 5–8 to DNA extracted from transfected parasites. Lanes 1 and 5 represent the detection of a portion of wild type locus (Pr19 and Pr20); lanes 2 and 6, the detection of wild type locus (Pr19 and Pr22); lanes 3 and 7 show the detection of episomal DNA (Pr25 and Pr26) and lanes 4 and 8 show the detection of the integration at the 5′end of the insert (Pr27 and Pr26). The absence of PCR product amplification using genomic DNA prepared from transfected parasite culture (Pr27 and Pr26) indicated the lack of homologous recombination (lane 8). D. PCR analysis of pCAM-PfI2-2HA transfected 3D7 culture (Knock-in strategy)). Lanes 1–4: DNA extracted from wild type parasites; lanes 5–8: DNA extracted from transfected parasites. Lanes 1 and 5: detection of a portion of wild type locus (Pr21 and Pr22); lanes 2 and 6: detection of wild type locus (Pr19 and Pr22); lanes 3 and 7: detection of episomal DNA (Pr25 and Pr28) and lanes 4 and 8: detection of the integration at the 3′end of the insert (Pr19 and Pr28). The amplification of a PCR product at ~ 1000 pb using genomic DNA prepared from transfected parasite culture (Pr19 and Pr28) indicated the homologous recombination and integration of the 2-HA construct in endogenous PfI2 (lane 8).
	Figure 4. Effect of PfI2 on PfPP1 phosphatase activity. A. Scheme of His-tagged recombinant wild-type, deleted and mutated PfI2 proteins used in this study. Recombinant PfPP1 was pre-incubated for 30 min at 37°C with different concentrations of PfI2WT (B), PfI2(1–94) (C), PfI2(19–144) (D), PfI2W16A (E) and PfI2Y103A (F) proteins before the addition of pNPP. Results presented as% of relative increase or decrease are means ± SEM of four independent experiments performed in duplicate.
	Figure 5. Study of PfI2-PfPP1 interaction and mapping of binding motifs in yeast two hybrid system. pGBKT7, pGBKT7-lam, pGBKT7-PfPP1 constructs were inserted into mat α (Y187) yeast and pGADT7, pGADT7-PfI2, pGADT7-PfI2(1–94), pGADT7-PfI2(19–144), pGADT7-PfI2W16A and pGADT7-PfI2Y103A constructs into mat a (AH109) yeast. These transformations were followed by mating (A). Yeast diploids were checked on SD-LW plates (B) and interactions were identified by growth on SD-LWH (C) or SD-LWHA (D). Western-blot analysis of extracts prepared from yeast diploids revealed with mAb anti-Gal4-AD (E) or mAb anti-Gal4-BD (F) showing the expected expression of the Gal4-AD tagged PfI2 proteins (PfI2WT: lanes 4,5,6; PfI2(1–94): lanes 7,8,9; PfI2(19–144): lanes 10,11,12; PfI2W16A: lanes 13,14,15 and PfI2Y103A: lanes 16,17,18) and Gal4-BD tagged laminin : lanes 2,5,8,11,14,17 or Gal4-BD tagged PfPP1: lanes 3,6,9,12,15,18.
	Figure 6. Initiation of G2/M transition in Xenopus oocytes by PfI2. Appearance of GVBD was monitored for 15 h after injection, and values are presented as percentages. Each experiment was performed using a set of 20 oocytes and was repeated at least three times. A. Percentage of GVBD induced with 100 ng of PfI2WT, PfI2(19–144), PfI2W16A or PfI2Y103A recombinant proteins. B. Immunoblot analysis of extracts prepared from injected oocytes revealed with mAb anti-His showing the presence of each recombinant protein after microinjection C. Interaction of PfI2 with Xenopus PP1. Immunoblot analysis using specific anti-XePP1 antibodies of naïve (lanes 1,4,7) or PfI2WT injected oocytes extracts after co-immunoprecipitation with anti-His mAb (lanes 3,6,9) (or anti-rabbit used as control (lanes 2,5,8)) revealed the presence of XePP1 in the complex (lanes 3,6). D. Percentage of GVBD induced by the pre-injection of PfI2(19–144), PfI2W16A or PfI2Y103A (100 ng) 2 hours before PfI2WT injection (100 ng). PfI2WT injection was used as a positive control.
	Figure 7. Role of synthetic peptides derived from PfI2. A. Schematic representation of PfI2 sequence and P1 and P4 peptides derived, respectively, from the RVxF (11KKTISW16) and HYNE (102HYNE105) domains. P6 (penetrating peptide) and P10 (P1 mutated version) were used as negative controls. B. Interaction studies of P1, P4, P6, P10 with His6-PfPP1 in vitro using ELISA based quantification of binding capacity. Increasing quantities of biotinylated His6-PfPP1 were added to wells coated with peptides (5 μg/well) or recombinant PfI2WT protein (5 μg/well) used as positive control. Results are representative of 4 independent experiments. C. Effect of P1, P4, P6 and P10 on PfPP1 phosphatase activity. Recombinant His6-PfPP1 at 27.03 nM (2.5 μg) was pre-incubated for 30 min at 37°C with different concentrations of peptides or PfI2WT (positive control) before the addition of pNPP. Results presented as % of relative increase or decrease are means ± SEM of four independent experiments performed in duplicate. D. Initiation of G2/M in Xenopus oocytes by peptides. Appearance of GVBD was monitored for 15 h after injection, and values are presented as percentages. Each experiment was performed using a set of 20 oocytes and was repeated at least three times. PfI2WT injection was used as positive control. E. Percentage of GVBD induced by the pre-injection of P1, P4, P6 or P10 (100 ng) 1 hour before PfI2WT injection (100 ng). PfI2WT injection was used as a positive control. F. Abolition of PfI2WT/XePP1 interaction after P1 and P4 peptides pre-injection. Extracts from oocytes pre-injected or not with peptides followed by PfI2WT injection were immunoprecipitated with anti-His mAb. Immunoblot analysis revealed with specific anti-XePP1 antibodies revealed the presence of XePP1 in the complex in P6 and P10 pre-injected extracts (lanes 4 and 5) and in non-pre-injected extracts (lane 1) but not in the P1 and P4 pre-injected extracts (lanes 2 and 3).
	Figure 8. Effect of synthetic peptides derived from PfI2 and PfI3 on blood P. falciparum parasite growth. A and B. Inhibition of growth of 3D7 P. falciparum strain parasites by PfI2 derived peptides. Parasites were incubated with different concentrations of synthetic peptides for 72 hr in a 96-well microtiter plate. Parasitemia was quantified as described in Methods. P1 and P4 correspond to KTISW and HYNE motifs respectively. P6 (penetrating peptide alone) and P10 (mutated version of P1) were used as control peptides. C. Schematic representation of PfI3 sequence (Pf Inhibitor 3): P5 peptide corresponds to 41KVVRW45 (RVxF motif) and P11 is the mutated version of P5 used as control peptide. D. Inhibition of growth of P. falciparum 3D7 by synthetic peptides derived from PfI3. Results presented as% of parasite growth inhibition are means ± SEM of seven independent experiments performed in duplicate. E. IC50 values of P1 and P5 against 3D7 and HB3 P. falciparum strains and against stimulated primary mouse spleen cells. F. Selective penetration of FITC-labeled P1 (F) and P5 (G) peptides in P. falciparum 3D7. The left panel represents the DAPI staining, the middle panel represents the fluorescence signal and the right panel represents the bright field image.

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