Figure S3 Effects of 2-aminoethyl diphenylborinate (2- APB) on mean amplitude of periodic calcium (Ca2+) fluctuations. A significant effect of 100 mM 2-APB (*, P = 0.0116, two-tailed unpaired t test) was detected only in merozoites. LRf, late ring forms; LT, late trophozoites; S, schizonts; M, merozoites. (TIF) Figure S4 Effects of U73122, thapsigargin (Tg) and concanamycin A (CMA) on Ca2+ oscillations in early ring forms (ERf) and early trophozoites (ET). (A) Spontaneous Ca2+ oscillations observed in ERf (left) and ET (right) disappeared after 5 min of pre-treatment with 10 mM U73122. (B, C) Erythrocytes were prepared in a manner identical to that for Ca2+ imaging in the culture chamber. Ca2+ imaging was initiated under the same conditions as those used for perfusion with normal culture medium. Replacement of perfusion medium with medium containing 2 mM thapsigargin (Tg) (B) or 100 nM concanamycin A (CMA) (C) induced an increase in Ca2+ concentration in ERf (open circles) and ET (closed squares). Black horizontal bars represent the period of perfusion with test compounds. (D) Ca2+ depletion induced by 30 min of pre- treatment with 2 mM Tg significantly diminished Ca2+ oscillations in ERf (left) and ET (right). (E) Ca2+ depletion induced by 30 min of pre-treatment with 100 nM CMA had no effect on Ca2+ oscillations in ERf (left) and ET (right). (TIF) Figure S5 Pre-treatment of erythrocytes with 2-APB did not inhibit intraerythrocytic development of P. falci- parum. To evaluate the effects of 2-APB on host cells, erythrocytes were pre-treated with 100 mM 2-APB for 1 h at 37uC, washed with RPMI medium and resuspended at a haematocrit of 5% in complete culture medium. Cultures of late schizonts (LS; 0.5% parasitaemia) were diluted four times with pre-treated erythrocytes, and the culture was continued. Cultures (three wells per experimental group) were terminated at 40 h of the assay, and thin smears of erythrocytes were prepared for parasite counting. Parasitaemia with ring forms did not differ significantly between dimethyl sulfoxide (DMSO)- and 2-APB-pre- treated groups (P = 0.408, two-tailed unpaired t test). Parasitaemia is shown as mean + S.D. of three independent counts of three wells. Stages with parasitaemia of less than 0.1% are not shown. (TIF) Figure S6 Effect of 2-APB on the area, perimeter and maximum diameter of the chloroquine-resistant strain K1. No statistically significant difference was observed in the area, perimeter and maximum diameter of intraerythrocytic parasites between DMSO- and 100 m M 2-APB-cultured groups after 24 h of the assay, but the 3 parameters showed a tendency to decrease. Error bars represent mean + S.D. (n = 50). P values are given in each panel (two-tailed unpaired t test). (TIF) References 1. World Health Organization (2011) World Malaria Report 2010. WHO. 2. Haldar K, Mohandas N (2007) Erythrocyte remodeling by malaria parasites. Curr Opin Hematol 14: 203–209. doi:10.1097/MOH.0b013e3280f31b2d. 3. Maier AG, Cooke BM, Cowman AF, Tilley L (2009) Malaria parasite proteins that remodel the host erythrocyte. Nat Rev Microbiol 7: 341–354. doi:10.1038/ nrmicro2110. 4. Dondorp AM, Pongponratn E, White NJ (2004) Reduced microcirculatory flow in severe falciparum malaria: pathophysiology and electron-microscopic pathology. Acta Trop 89: 309–317. Plasmodium Ca2+ Oscillation in the Blood Stage Figure S7 Timetable of the effects of 2-APB on Ca2+ dynamics and parasite development in the blood stage of P. falciparum. Images show Giemsa-stained parasites. Blue bars above the parasite images indicate stages of the parasites in which spontaneous Ca2+ oscillations or small periodic Ca2+ fluctuations were observed. Red lines represent the period of 2- APB treatment. Red circles indicate the initiation of 2-APB treatment. Black, dark and light grey boxes show the extent of the effect of 2-APB during each sampling period: black, severe effect with developmental delay and abnormal morphology; dark grey, weaker effect with developmental delay and abnormal morphol- ogy than that when parasites were exposed to 2-APB for 40 h; light grey, slight effect with developmental delay or abnormal morphology compared to that when parasites were exposed to 2- APB for 40 h. (TIF) Figure S8 Electron micrographs of parasites in DMSO control culture. (A) At 30 h of the assay (original magnification, 630,000). (B) Higher magnification image of the part marked with an asterisk in (A) (original magnification, 680,000). (C) At 30 h of the assay (original magnification, 620,000). (D) Higher magnifi- cation image of the part marked with an asterisk in (C) (original magnification, 680,000). N, nucleus; NE, nuclear envelope; Ri, ribosome; Rh, rhoptry. (TIF) Figure S9 Effects of 2-APB on the number of merozoites in each schizont. (A) Box plot of the number of merozoites (M) formed in each schizont (S) after 40 h of culture with DMSO (white boxes) or 2-APB (light grey boxes). The central rectangle spans the first quartile to the third quartile. The segment inside the rectangle shows the median and whiskers above and below the box show the minimum and maximum. 2-APB significantly decreased the number of M per S (two-tailed unpaired t test with Welch’s correction). (B) Frequency distribution of the raw data shown in (A). (TIF) Acknowledgments We thank Dr S. Kano (National Center for Global Health and Medicine) for providing the P. falciparum strains, the Hokkaido Kushiro Red Cross Blood Center for supplying human red blood cells, Dr S. Harakawa and Dr M. Asada (National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine) for technical help in fluorescence Ca2+ imaging and ER staining and Ms F. Yokotsuka (Institute for Medical Science, Dokkyo Medical University) for technical help in the measurement of parasite size. Author Contributions Conceived and designed the experiments: ME S. Kawazu S. Kawai KM. Performed the experiments: ME S. Kawazu S. Kawai WF TI JW. Analyzed the data: ME S. Kawazu S. Kawai WF. Wrote the paper: ME S. Kawazu KM. { Deceased 5. Berridge MJ, Lipp P, Bootman MD (2000) The versatility and universality of calcium signalling. Nat Rev Mol Cell Biol 1: 11–21. doi:10.1038/35036035. 6. Clapham DE (2007) Calcium signaling. Cell 131: 1047–1058. doi:10.1016/ j.cell.2007.11.028. 7. Nagamune K, Hicks LM, Fux B, Brossier F, Chini EN, et al. (2008) Abscisic acid controls calcium-dependent egress and development in Toxoplasma gondii. Nature 451: 207–210. doi:10.1038/nature06478. PLoS ONE | www.plosone.org 11 July 2012 | Volume 7 | Issue 7 | e39499