Additional file 2: of Monoclonal antibody therapy for Kawasaki disease: a protocol for systematic reviews and meta-analysis

Search terms and strategies. The search strategy utilized is outlined in more detail in the file. (DOCX 41 kb

Additional file 1: of Monoclonal antibody therapy for Kawasaki disease: a protocol for systematic reviews and meta-analysis

PRISMA-P checklist: recommended items to address in a systematic review protocol. (DOC 81 kb

Genetic characteristics of genome donors and selection of the trans-mitochondrial cybrids.

a<p>ρ⁰ MDA-MB-231 cells were used as nuclear DNA donors and mtDNA recipients. As mtDNA donors, we used HeLamt231 and HeLamtFt cybrids sharing the HeLa nuclear genome background to exclude the influence of variations in nuclear-coded cytoplasmic factors on the phenotypes. HeLamt231 cybrids carrying nuclear DNA from HeLa cells and mtDNA from MDA-MB-231 cells were isolated by the fusion of ρ∼ HeLa cells with enucleated MDA-MB-231 cells and subsequent cultivation in selection medium with 6-thioguanine medium without uridine and pyruvate (6-tg+UP⁻). The 6-tg eliminates unenucleated MDA-MB-231 cells and UP⁻ eliminates unfused ρ⁰ HeLa cells, which require uridine and pyruvate due to their complete respiration defects caused by mtDNA depletion. HeLamtFt cybrids carrying nuclear DNA from HeLa cells and mtDNA from human fetal skin fibroblasts were obtained previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023401#pone.0023401-Hayashi4" target="_blank">[17]</a> by the fusion of ρ⁰ HeLa cells with enucleated human fetal skin fibroblasts and subsequent cultivation in the 6-tg+UP⁻ medium.</p>b<p>en represents enucleated.</p>c<p>The mtDNA donors sharing the HeLa nuclear genome background and expressing 6-tg resistance cannot survive in the presence of hypoxanthine/aminopterin/thymidine (HAT). Nuclear donor ρ⁰ MDA-MB-231 cells can grow in the HAT selection medium, but not in UP^- selection medium. Thus, only transmitochondrial cybrids can survive in the selection medium.</p

Genotyping of mtDNA in the isolated transmitochondrial cybrids.

<p>The ρ⁰ MDA-MB-231 cells have had their mtDNA removed and were used as mtDNA recipients and nuclear DNA donors; HeLamt231 and HeLamtFt correspond to mtDNA donors sharing the HeLa nuclear genome, but possessing mtDNA from the MDA-MB-231 cells and human fetal fibroblasts, respectively (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023401#pone-0023401-t002" target="_blank">Table 2</a>). For identification of the C12084T mutation exclusively present in mtDNA of MDA-MB-231 cells (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023401#pone-0023401-t001" target="_blank">Table 1</a>), the PCR products were digested with <i>Ear</i>I. Because of the gain of an <i>Ear</i>I site due to the C12084T mutation, 231mt231 cybrids carrying mtDNA from MDA-MB-231 cells produce 113- and 33-bp fragments, whereas 231mtFt cybrids carrying normal mtDNA without the mutation produce a 146-bp fragment.</p

Expression of nuclear-coded genes related to metastasis in the isolated transmitochondrial cybrids.

<p>Normal mtDNA from fetal fibroblasts was transferred into ρ⁰ MDA-MB-231 cells, and its effects on the expression of the nuclear-coded genes related to metastasis were examined using real-time PCR analysis (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023401#s2" target="_blank">Materials and Methods</a>). Bars represent the mean ± S.D. (<i>n</i> = 3).</p

Characterization of human non-metastatic MCF-7 and highly metastatic MDA-MB-231 breast carcinoma cells.

<p>(<b>A</b>) Experimental metastatic potential. Numbers of lung nodules were counted after inoculation of the cells into the tail vein of nu/nu mice. (<b>B</b>) Biochemical analysis of mitochondrial respiratory complex activities. Respiratory complex I, complex II, complex III, and complex IV are components of the electron-transport chain and are located in the mitochondrial inner membrane. Mitochondrial respiratory function was examined by estimating the activities of the complexes. Because the activity of complexes II+III is comparable in MCF-7 and MDA-MB-231 cells, the reduced activity of complexes I+III in MDA-MB-231 cells must result from complex I defects. Bars represent the mean ± S.D. (<i>n</i> = 3). *<i>P</i><0.05; **<i>P</i><0.01. (<b>C</b>) Estimation of the amounts of ROS. Flow-cytometric analysis was carried out using 1×10⁶ cells, which were treated with 5 µM DCFH-DA for quantitative estimation of ROS production.</p

Characterization of the phenotypes of the isolated transmitochondrial cybrids.

<p>Using 231mtFt and 231mt231 cybrids, we examined the effects of transferring normal mtDNA from fetal fibroblasts into ρ⁰ MDA-MB-231 on (<b>A</b>) mitochondrial respiratory function, (<b>B</b>) lactate production, (<b>C</b>) ROS production, and (<b>D</b>) experimental metastatic potential. Bars represent the mean ± S.D. (<i>n</i> = 3). *<i>P</i><0.05; **<i>P</i><0.01.</p