Primers used in this study.

a<p>“-S” and “-As”: indicate the sense (-S) and antisense (-As) primers of the corresponding genes.</p><p>b “-f” and “-r”: indicate the forward (-f) and reverse (-r) primers used in the site-directed mutagenesis.</p><p>Underlined bases indicate the mutant sites.</p><p>Primers used in this study.</p

GC/TOF-MS identification of the products generated by YlIDH R141H and ScIDH1 R148H catalysis.

<p>(A) and (D) Gas chromatograms of the products generated by YlIDH R141Hand ScIDH1 R148H catalysis, respectively. (B) and (E) Mass spectrogram identifications of the 11.94-min peak in (A) and 11.91-minpeak in (D), respectively.(C) and (F) Mass spectrogram identifications of the 17.43-min peak in (A) and (D), respectively.</p

GC/TOF-MS identification of the 2-HG, ICT and α-KG standards.

<p>The retention times of the 2-HG, ICT and α-KG standards in the gas chromatogram were 11.94 min (A), 17.44 min (B) and 12.03 min (C), respectively. (D), (E) and (F) show the mass spectrogram identifications of 2-HG, ICT and α-KG, respectively.</p

Comparison of the kinetic parameters of the isocitrate oxidation activity of the wild-type and mutant IDH enzymes.

a<p>When calculating the <i>K</i>_m for isocitrate, the concentration of D-isocitrate was calculated as 50% of the total DL-isocitrate in this study. “na” indicates no measurable activity.</p><p>Comparison of the kinetic parameters of the isocitrate oxidation activity of the wild-type and mutant IDH enzymes.</p

Circular dichroism (CD) spectra analysis of intact and mutant YlIDH, ScIDH1 and HcIDH.

<p>The CD was measured, and the molar ellipticity was calculated as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115025#s2" target="_blank">Materials and Methods</a>. (A) The molar ellipticities of HcIDH (▪), HcIDH R148H (•), HcIDH R132A (▴) and HcIDH R132E (▾) from 195 to 260 nm; (B) the molar ellipticities of ScIDH1 (▪), ScIDH1 R148H (•), ScIDH1 R148A (▴) and ScIDH1 R148E (▾) from 195 to 260 nm; (C) the molar ellipticities of YlIDH (▪), YlIDH R141H (•), YlIDH R141A (▴) and YlIDH R148E (▾) from 195 to 260 nm.</p

SDS-PAGE analysis of the expression and purification of wild-type and mutant YlIDH, ScIDH1 and HcIDH.

<p>Analysis was performed using a 12% polyacrylamide gel. M, protein molecular weight markers; lane 1 to 4, purified proteins of YlIDH, YlIDH R141H, YlIDH R141A and YlIDH R141E, respectively; lane 5 to 8, purified proteins of ScIDH, ScIDH R148H, ScIDH R148A and ScIDH R148E, respectively; lane 9 to 12, purified proteins of HcIDH, HcIDH R132H, HcIDH R132A and HcIDH R132E, respectively.</p

Structure-based protein sequence alignment of ScIDH1, YlIDH and HcIDH.

<p>The conserved amino acid residues are shaded. The secondary structure components, j.e, α-helices and β-sheets, of HcIDH and ScIDH1 are also shown. The conserved amino acid residues involved in substrate binding (★) and cofactor binding (▴) are indicated. This figure was generated using ESPript 2.2.</p

Comparison of the kinetic parameters for the α-KG reduction activity of the mutant IDHs.

<p>Comparison of the kinetic parameters for the α-KG reduction activity of the mutant IDHs.</p

Potential involvement of the 18 kDa translocator protein and reactive oxygen species in apoptosis of THP-1 macrophages induced by sonodynamic therapy

<div><p>Sonodynamic therapy (SDT) with exogenous protoporphyrin IX (PpIX) or endogenous PpIX derived from 5-aminolevulinic acid (ALA) has been carried out to produce apoptotic effects on macrophages, indicating a potential treatment methodology for atherosclerosis. Our previous studies have found that mitochondria damage by reactive oxygen species (ROS) plays a major role in the SDT-induced apoptosis. This study aimed at investigating the potential involvement of the mitochondrial 18 kDa translocator protein (TSPO) and ROS in the pro-apoptotic effects of SDT on THP-1 macrophages. THP-1 macrophages were divided into control and SDT groups, and went through pretreatment of the specific TSPO ligand PK11195 and ROS scavengers N-Acetyl Cysteine (NAC), then compared with groups without pretreatment. Application of PK11195 reduced intracellular accumulation of endogenous PpIX. PK11195 and NAC reduced the generation of intracellular ROS and oxidation of cardiolipin induced by SDT, respectively. PK11195 and NAC also reduced SDT-induced mitochondrial membrane potential (ΔΨ_m) loss, the translocation of cytochrome c and cell apoptosis. PpIX accumulation, ROS generation and cell apoptosis were also attenuated by siTSPO. Our findings indicate the pivotal role of TSPO and ROS in SDT-induced cardiolipin oxidation, ΔΨ_m collapse, cytochrome c translocation and apoptosis in THP-1 macrophages.</p></div

Effects of PK 11195 and NAC on SDT-induced macrophage apoptosis.

<p>THP-1 macrophages were treated for 3 hours with 5-aminolevulinic acid mediated sonodynamic therapy (ALA-SDT), with or without pretreatment of PK 11195 and NAC. Cell apoptosis and necrosis were assessed by flow cytometry with double staining of Annexin V and propidium iodide (PI). (A) Cells in the lower-right quadrant (Annexin-V⁺/PI^-) represent early apoptotic cells. (B) Quantifications of early apoptosis rate in the indicated groups. ***<i>P</i> < 0.001 compared to no treatment group, ^##<i>P</i> < 0.01 compared to ALA-SDT treated group, ^###<i>P</i> < 0.001 compared to ALA-SDT treated group.</p