Photobleaching decay curves of GFP from 3D7-KAHRP(+His)-GFP-TC parasites, and ReAsH and FlAsH from 3D7-KAHRP(+His)-TC labeled parasites in <i>Pf-</i>infected erythrocytes.

<p>Continuous exposure of cells for 20 s to the 9.98 mW 475 nm±20 nm light (for GFP and FlAsH), or to the 10.88 mW 560 nm±22 nm light (for ReAsH) cause fluorescence decays to 65%, 73%, and 85% of the initial emission signals from GFP, FlAsH-, and ReAsH-labeled TC tagged protein, respectively.</p

Transfection plasmids and detection of GFP fusion proteins.

<p>(A) Plasmid pHH2-KAHRP(+His)-GFP <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022975#pone.0022975-Wickham1" target="_blank">[14]</a> was used to amplify the first 60 amino acids of KAHRP containing the putative hydrophobic signal sequence followed by the histidine rich region with or without GFP fusion. <i>3′hrp2</i>, histidine-rich protein-2 3′UTR; <i>hDHFR</i>, human dihydrofolate reductase gene; <i>5′CAM</i>, <i>Pf</i> calmodulin promoter region; <i>5′hsp86</i>, heat-shock protein-86 promoter region; <i>3′PcDT</i>, <i>P. chabaudi</i> dihydrofolate reductase 3′UTR; Fp1-KAHRP, forward primer used to add a <i>Spe</i>I restriction site (Sp) immediately before the first codon of KAHRP cloned into pDC-pvcrt-o-MH (not shown); Rp1-GFP-TC, reverse primer used to amplify GFP, add an <i>Eco</i>RV restriction site (Ev) in frame between GFP and the TC tag (for subcloning of other genes), and add a <i>Xma</i>I restriction site (Xm) after the TC sequence, for cloning into pDC-pvcrt-o-MH; Rp2-His-TC, reverse primer used to amplify the histidine rich region of KAHRP, add an <i>Eco</i>RV restriction site (Ev) while maintaining the reading frame of KAHRP(+His) and the TC tag (for future subcloning of other genes), and add a <i>Xma</i>I restriction site (Xm) after the TC sequence, for cloning into pDC-pvcrt-o-MH. (B) Transfection plasmid pDC-KHT contains the sequence for the fusion protein KAHRP(+His)-TC from pHH2-KARHP(+His)-TC. Myc (M) and His×6 (H) tags are encoded immediately after the <i>Xma</i>I restriction site. (C) Transfection plasmid pDC-KHGT contains the sequence of KAHRP(+His)-GFP-TC in frame with codons for a PRGTKTYF terminus (F) that begin at the <i>Xma</i>I site. <i>5′PcDT</i>, <i>P. chabaudi</i> dihydrofolate reductase promoter region; <i>3′hsp86</i>, heat-shock protein-86 3′UTR. (D) Immunoblot image shows antibody detection of GFP as a M<i>_r</i> 28,000 band, detection of KAHRP(+His)-GFP-TC as a M<i>_r</i> 42,000 band, and detection of M<i>_r</i> 39,000 and M<i>_r</i> 29,000 bands from KAHRP(+His)-GFP protein. No band was detected from control non-transformed 3D7 PE.</p

S/N ratios of GFP, FlAsH and ReAsH fluorescence from 3D7-KAHRP(+His)-GFP-TC- and 3D7-KAHRP(+His)-TC-transformed PE.

<p>S/N ratios of GFP, FlAsH and ReAsH fluorescence from 3D7-KAHRP(+His)-GFP-TC- and 3D7-KAHRP(+His)-TC-transformed PE.</p

Measurements of cell multiplication and 50% and 90% inhibitory concentrations (IC₅₀; IC₉₀).

<p>Values are shown as mean of 3 experiments in duplicate with standard errors. Parasites lines 3D7, 3D7-KAHRP (+His)-GFP-TC and 3D7-KAHRP (+His)-TC were exposed for 15 min, and 1 h to different concentrations of BAL (0 mmol/L–5 mmol/L).</p

KAHRP protein trafficking in PEs.

<p>(A–C) Bright field (BF) and GFP fluorescence images of PEs containing <i>Pf</i> 3D7 parasites transformed to express KAHRP(+His)-GFP protein without TC tag. (D–F) Images of 3D7-KAHRP(+His)-GFP-TC PE labeled with ReAsH and photographed in the green and red channels separately for GFP and ReAsH fluorescence. (G–I) Images of 3D7-KAHRP(+His)-TC PE labeled with FlAsH. (J–L) Images of 3D7-KAHRP(+His)-TC PE labeled with ReAsH. The emission crossover between green and red channels was negligible, and was estimated to be less than a 1% leak of GFP emission into the ReAsH emission channel. Scale bar represents 5 µm.</p

Fluorescence microscopy images of ReAsH and FlAsH labeled PEs.

<p>(A) Bright field (BF) and fluorescence images of non-transformed 3D7 PE labeled with ReAsH, not treated with BAL. (B) BF and fluorescence images of non- transformed 3D7 PE labeled with FlAsH, not treated with BAL. (C) BF and fluorescence images of 3D7 PE treated with BAL and labeled with ReAsH. (D) BF and fluorescence images of 3D7 PE treated with BAL and labeled with FlAsH. (E) BF and fluorescence images of 3D7-KAHRP(+His)-TC PE treated with BAL and labeled with ReAsH. (F) BF and fluorescence images of 3D7-KAHRP(+His)-TC PE treated with BAL and labeled with FlAsH. (G) BF and fluorescence images of 3D7-KAHRP(+His)-GFP-TC PE treated with BAL and labeled with ReAsH. (H) Images of non- transformed 3D7 PE exposed first to 200 µmol/L CPM and followed by labeling with 2.5 µmol/L ReAsH. Excitation and imaging of ReAsH, FlAsH and CPM fluorescence were performed with wavelengths and filter sets described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022975#s2" target="_blank">Materials and Methods</a>. Scale bar represents 10 µm.</p

Detergent-resistant membrane analyses of AA and CC erythrocytes.

<p>Proteins from 18 fractions (out of a total of 36 fractions) were separated by SDS-PAGE under denaturing conditions, transferred to PVDF membrane, and probed with protein-specific monoclonal antibodies.</p

Zeta potential (ZP) analyses.

<p>(A) Diagram of ZP principle. ZP is defined as the electrochemical potential at the shear plane. Outside the shear plane, ions are not closely associated with the internal ion cloud. (B) ZP measurements from AA and CC erythrocyte populations. Peak values were estimated by Gaussian fitting the histogram. (C) Levels of membrane-associated hemichromes (mean±SD) in control and NaNO₂-treated AA erythrocytes. For reference, native CC erythrocytes show 1.8-fold greater hemichrome levels than native AA erythrocytes <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005828#pone.0005828-Fairhurst1" target="_blank">[31]</a>. (D) ZP measurements from control and NaNO₂-treated AA erythrocyte populations.</p

Flow cytometry analyses of phosphatidylserine on the surface of unfixed AA and CC erythrocytes.

<p>Erythrocytes were reacted with FITC-labeled Annexin-V. Subpopulations of AA (A) and CC (B) erythrocytes were gated and the percentage of total erythrocytes within each gate was calculated. Normalized mean fluorescence intensities of the low and high side scatter populations (black and red arrows, respectively) are shown in panel C. Error bars represent SEM (n = 4).</p

Distributions of relative band 3 and CD47 signals from dot blot analyses.

<p>For each protein, the sum of signals from all fractions was set as 100%. Band 3 (A, B) and CD47 (C, D) signals showed similar distributions in the high density fractions of AA and CC samples. However, differences in band 3 and CD47 signal distributions were observed in the low density fractions of AA and CC samples. Higher density signals for both proteins in CC samples appeared in fractions 9–11, which correspond to visible DRM.</p