Optimization of the medium for amastigote-to-trypomastigote transformation.

<p>(A) Amastigotes (1 × 10⁶ cells/mL) were cultured in Grace’s Insect Medium, RPMI-1640 medium, 80% RPMI-1640 plus 20% Grace’s insect medium, or 70% RPMI-1640 plus 20% Grace’s insect medium containing 10% FBS for 7 days. The histogram depicts the percentage of intermediate forms and trypomastigotes, which was calculated by daily counting of the different forms by using IX71 with 60 x objective. More than 100 parasites were randomly counted. (B) The percentage of trypomastigotes is shown in Fig 2A. Data shown are the mean ± S.D. of 3 independent experiments. *<i>P</i> < 0.05, **<i>P</i> < 0.01.</p

Additional file 4: Figure S3. of Attenuation of teratoma formation by p27 overexpression in induced pluripotent stem cells

Expression profile of stem cell factors in miPSCs-p27. RT-PCR analysis of stem cell marker genes (A) and relative intensities (B) of miPSCs and miPSCs-p27 are shown. Error bars correspond to the SEM (n = 3). *P < 0.05, student’s t-test. (PDF 311 kb

Involvement of TcIP₃R in amastigote-to-trypomastigote transformation.

<p>(A) Amastigotes of WT or <i>TcIP</i>_<i>3</i><i>R</i>-SKO- or EGFP-TcIP₃R-overexpressing parasites were cultured in a medium composed of 70% RPMI-1640 and 20% Grace’s Insect Medium supplemented with 10% FCS for 7 days. The percentage of the intermediate forms plus trypomastigotes was calculated daily. (B) The percentage of trypomastigotes is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135726#pone.0135726.g003" target="_blank">Fig 3A</a>. Data shown are the mean ± S.D. of 3 independent experiments. *<i>P</i> < 0.05, **<i>P</i> < 0.01.</p

Additional file 3: Figure S2. of Attenuation of teratoma formation by p27 overexpression in induced pluripotent stem cells

Effects of p27 overexpression on EB formation. Percentages of wells which have EBs and beating EBs are shown in A and B, respectively. Error bars correspond to the SEM (n = 3). *P < 0.05, student’s t-test. (PDF 341 kb

Identification of PLC isozymes involved in IP₃ generation in HeLa cells stimulated with histamine.

<p>(A) Representative traces of IRIS-1 signal changes (ΔR/R₀; top) and Indo-5F signal changes (F/F₀; bottom) observed in thapsigargin-treated HeLa cells. The horizontal broken lines indicate the baseline levels. (B) Traces of the mean ± SD IRIS-1 signal changes (ΔR/R₀) observed in thapsigargin-treated HeLa cells after addition of 3 µM histamine (blue circles) or 3 µM histamine plus 2 mM Ca²⁺ (green circles). (C–F) Effects of PLC isozyme knockdown on the IP₃ increase evoked by 3 µM histamine alone (C), the IP₃ increase evoked by 2 mM Ca²⁺ alone (D), the first component of the IP₃ increase evoked by 3 µM histamine plus 2 mM Ca²⁺, and the second component of the IP₃ increase evoked by 3 µM histamine plus 2 mM Ca²⁺. Data are shown as means ± SD. The numbers of cells measured are shown in parentheses. Statistical analyses were performed by one-way ANOVA followed by Scheffe’s multiple comparison test. *P<0.05, **P<0.01, vs. the values in control siRNA-transfected cells.</p

Additional file 1: Table S1. of Attenuation of teratoma formation by p27 overexpression in induced pluripotent stem cells

Specific primer sequences for RT-PCR analyses. (DOCX 12 kb

Effects of PLC-β1 or PLC-β4 overexpression on Ca²⁺ and IP₃ dynamics evoked by histamine stimulation.

<p>(A) Western blotting analyses of cell lysates prepared from HeLa cells transfected with PLC-β1-IRES-mRFP (lane 2) or PLC-β4-IRES-mRFP (lane 4). Non-transfected cells were used as controls (lanes 1 and 3). (B) Representative traces of Indo-5F signal changes (F/F₀; top) and IRIS-1 signal changes (ΔR/R₀; bottom) in transfected cells stimulated with 3 µM histamine. The plasmid DNAs used to transfect the cells are shown on the left. The horizontal broken lines indicate the baseline levels of IRIS-1 and Indo-5F signals. The vertical broken lines indicate the onsets of stimulation. (B–D) Histograms for the inverse time constants for exponential decay of the Ca²⁺ oscillation amplitude (B), Ca²⁺ oscillation frequencies (C), and integrated IP₃ signals (D) in cells expressing mRFP (top row), PLC-β1 and mRFP (second row), and PLC-β4 and mRFP (third row). The means ± SD are shown at the bottom. The numbers of cells measured are shown in parentheses. Statistical analyses were performed by one-way ANOVA followed by Scheffe’s multiple comparison test. **P<0.01, vs. the values in IRES-mRFP transfected cells.</p

A possible mechanism that underlies the generation of cell-specific patterns of histamine-induced Ca²⁺ signals.

<p>The diagrams show the typical IP₃ and Ca²⁺ dynamics observed in HeLa cells stimulated with histamine. Low-frequency sustained Ca²⁺ oscillations tend to be observed in cells in which PLC-β1 is dominant (left), while high-frequency damped oscillations tend to be observed in cells in which PLC-β4 is dominant (right).</p

Biological properties of trypomastigotes derived from <i>in vitro</i> trypomastigogenesis.

<p>(A) Tissue-culture trypomastigotes (a), metacyclic trypomastigotes (b), amastigotes (c), or the parasites cultured for 6 d in trypomastigogenesis medium (d) were fixed, incubated with anti-<i>trans</i>-sialidase antibody, and stained with Alexa Fluor 488-labelled (green) secondary antibody. The nuclei (n) and kinetoplast (k) (blue) were counter-stained using Hoechst 33342. (B) Trypomastigotes derived from trypomastigogenesis or tissue-culture trypomastigotes (2 × 10⁵) were incubated with 2 × 10⁴ 3T3-Swiss albino cells for 12 h. For calculation of the infectivity, the number of intracellular parasites in a total of 200 cells was counted after Giemsa staining. Data shown are the mean ± S.D. of 3 independent experiments. Statistical analysis between the groups was performed using Student’s <i>t</i>-test.</p

siRNA sequences.

a<p>The GC% column indicates the GC content of each siRNA sequence.</p>b<p>The numbers in the position column denote where the sequence is located in the mRNA of the target gene (counted from the first nucleotide of the start codon) of human origin.</p