The effect of TRAP1 on the expression levels of mitochondrial fusion proteins.

<p>A, Cells were fractionated into cytoplasm (cyto) and mitochondrial (mito) fractions, which were analyzed by western blotting using antibodies against mitofusin (Mfn), OPA1, TRAP1, HADHA (mitochondrial marker) and β-tubulin (cytoplasmic marker). B, Quantification of the expression levels of Mfn1/2 and OPA1. These data were obtained by densitometric analysis of each band on the western blots, and the data are expressed as the means ± s.e.m. of at least four independent experiments. β-tubulin (cyto) and HADHA (mito) were used as internal controls. One-way ANOVA showed no statistically significant difference between the groups (Mfn1, <i>P</i> = 0.414; Mfn2, <i>P</i> = 0.636; OPA1, <i>P</i> = 0.093).</p

The effect of transient TRAP1 KD on mitochondrial morphology in SH-SY5Y cells.

<p>A, Cells were analyzed by immunofluorescence microscopy using anti-HADHA antibody. High magnification images of a part of each photograph are shown in the white windows (normal, untreated; control, scrambled siRNA; siRNA, siRNA to TRAP1). B, Percentages of cells with the indicated mitochondrial morphologies in normal SH-SY5Y cells, control and TRAP1 KD cells. C, Cells were fractionated into mitochondrial (mito) fractions and blotted with antibodies against TRAP1, Drp1, Mff, fis1, Mfn1/2, OPA1, HADHA and β-tubulin. D, Quantification of the results shown in panel C. Data are expressed as the means ± s.e.m. of at least three independent experiments. The conventions are the same as those shown in Fig. 3. The asterisks on each bar indicate statistical significance (TRAP1: <i>P</i> = 0.025, Drp1: <i>P</i> = 0.012, Mff: <i>P</i> = 0.001, Fis1: <i>P</i> = 0.614, Mfn1: <i>P</i> = 0.764, Mfn2: <i>P</i> = 0.432, OPA1: <i>P</i> = 0.343, vs. control, Welch’s t-test, *<i>P</i><0.05, **<i>P</i><0.01). Scale bar, 10 µm (A).</p

Localization of TRAP1 in SH-SY5Y cells.

<p>A, SH-SY5Y cells were stained with anti-TRAP1 and anti-HADHA antibodies (mitochondrial marker) (a,d, TRAP1, green; b,e, HADHA, red; c,f, overlapping, yellow) and observed under a confocal microscope as described in the Materials and Methods. Upper panels show low magnification images. Lower panels show higher magnification images of part of each low magnification image (white windows). B, Equal amounts of lysates from cytoplasm (cyto) and mitochondria (mito) were analyzed by western blotting using anti-TRAP1 (upper panels), anti-HADHA (middle panel) or β-tubulin antibodies (lower panels). Scale bars, 20 µm (A, upper), 10 µm (A, lower).</p

MG132 treatment affects the expression levels of Drp1 and Mff in TRAP1 KD cells.

<p>A, Quantitative RT-PCR of Drp1 and Mff mRNA levels in TRAP1 knockdown cells. Actin mRNA was used as an internal control. Data represent the means ± s.e.m. (Drp1: <i>P</i> = 0.863; Mff: <i>P</i> = 0.975, vs. control, Welch’s t-test) (n = 3). B, Cells treated with 1 µM MG132 were fractionated into mitochondrial (mito) fractions and blotted with antibodies against TRAP1, Drp1, Mff, HADHA and β-tubulin. C, Quantification of the results shown in panel B. The data are expressed as the means ± s.e.m. of at least six independent experiments. The asterisks on each bar indicate statistically significant differences between the group and the normal SH-SY5Y cells (Drp1: one-way ANOVA, <i>P</i> = 2.19×10⁻⁵, post hoc Games-Howell test, *<i>P</i><0.05; Mff: one-way ANOVA, <i>P</i> = 1.94×10⁻⁴, post hoc Games-Howell test, *<i>P</i><0.05). D, Cells were fractionated into mitochondrial (mito) fractions and blotted with antibodies against TRAP1, Parkin, MarchV, HADHA and β-tubulin. E, Quantification of the results shown in panel D. Data represent the means ± s.e.m. (Parkin: <i>P</i> = 0.159; Mff: <i>P</i> = 0.994, vs. control, Welch’s t-test) (Parkin: n = 8; MarchV: n = 3).</p

The effect of Drp1 and Mff overexpression on mitochondrial morphology.

<p>A, Mitochondrial fractions (mito) from cells infected with GFP, Mff291, Mff342 or HA-Drp1 were blotted with antibodies against TRAP1, Drp1, Mff, HADHA and β-tubulin. B, Cells treated as in A were analyzed by immunofluorescence microscopy using anti-HADHA antibody. C, Percentages of cells with the indicated mitochondrial morphology in B. Data were collected from three independent experiments and represent means ± s.e.m. Scale bar, 10 µm (B).</p

The effect of KD on TRAP1 expression and mitochondrial morphology.

<p>A, Specific reduction of TRAP1 levels in the clone stably expressing human TRAP1 shRNA, as shown by western blotting. Total proteins isolated from normal SH-SY5Y cells, control shRNA-expressing cells (cont) and the TRAP1 shRNA-expressing SH-SY5Y cells (TRAP1 KD) were blotted using antibodies against TRAP1 and β-tubulin. B, Cells were stained with an antibody against the mitochondrial marker, HADHA. High magnification images of a part of each photograph are shown in the white windows. C, Percentages of cells with the indicated mitochondrial morphologies in normal SH-SY5Y cells, control and TRAP1 KD cells. Data were obtained from at least three independent experiments and represent the means ± s.e.m. D, Representative electron micrographs of control and stable TRAP1 KD cells. Arrows indicate examples of fragmented mitochondria, and arrowheads indicate examples of tubular mitochondria. E, Quantification of mitochondrial length in each cell. The graph on the left shows the length of the mitochondrial long axis. The graph on the right shows the ratio of the lengths of the mitochondrial long axis and short axis (aspect ratio). The data are expressed as the means ± s.e.m. of 126 mitochondria (13 cells, control), 175 mitochondria (11 cells, KD #1), and 117 mitochondria (11 cells, KD #2). The asterisks on each bar indicate statistical significance among the groups (long axis: one-way ANOVA, <i>P</i> = 9.06×10⁻²⁰, post hoc Games-Howell test, ***<i>P</i><0.001; aspect ratio: one-way ANOVA, <i>P</i> = 1.55×10⁻¹⁷, post hoc Games-Howell test, ***<i>P</i><0.001). Scale bars, 10 µm (B), 1 µm (D).</p

Transient TRAP1 KD induces abnormal mitochondrial morphology and a strong decrease in Drp1 level in KNS cells.

<p>A, Cells were stained with an anti-HADHA antibody and analyzed by immunofluorescence microscopy. High magnification images of a part of each photograph are shown in the white windows (control, scrambled siRNA; siRNA, siRNA to TRAP1). B, Percentages of cells with the indicated mitochondrial morphologies in control and TRAP1 KD cells. C, Mitochondrial fractions (mito) from each cell group were blotted with antibodies against TRAP1, Drp1, Mff, fis1, Mfn1/2, OPA1, HADHA and β-tubulin. D, Quantification of the results shown in panel C. The data are expressed as the means ± s.e.m. of at least three independent experiments. The conventions are the same as those shown in Fig. 3. The asterisks on each bar indicate statistical significance (TRAP1: <i>P</i> = 0.030, Drp1: <i>P</i> = 0.778, Mff: <i>P</i> = 0.030, Fis1: <i>P</i> = 0.355, Mfn1: <i>P</i> = 0.286, Mfn2: <i>P</i> = 0.173, OPA1: <i>P</i> = 0.199, vs. control, Welch’s t-test, *<i>P</i><0.05). Scale bar, 20 µm (A).</p

The effect of TRAP1 on the expression levels of mitochondrial fission proteins.

<p>A, A, The expression levels of Drp1, Mff and Fis1 were analyzed by western blotting. B, Quantification of the expression levels of Drp1, Mff and Fis1. The conventions are the same as those shown in Fig. 3. The asterisks on each bar indicate statistical significance between the group and normal SH-SY5Y cells (Drp1 (cyto): one-way ANOVA, <i>P</i> = 2.02×10⁻⁵, post hoc Games-Howell test, *<i>P</i><0.05, **<i>P</i><0.01; Mff: one-way ANOVA, <i>P</i> = 1.53×10⁻⁶, post hoc Games-Howell test, *<i>P</i><0.05, **<i>P</i><0.01; Fis1: one-way ANOVA, <i>P</i> = 0.684). C, Mitochondrial fractions from cells were blotted with antibodies against TRAP1, MiD51/MIEF1, HADHA and β-tubulin. D, Quantification of the expression level of MiD51/MIEF1. Data are expressed as the means ± s.e.m. of seven independent experiments. Welch’s t-test showed no statistical significance (<i>P</i> = 0.863).</p

Overexpression of TRAP1 affects mitochondrial morphology and the expression level of mitochondrial fission proteins.

<p>A, Cells infected with the GFP or TRAP1-GFP adenoviruses were fractionated into cytoplasmic (cyto) and mitochondrial (mito) fractions and blotted with antibodies against TRAP1, Drp1, Mff, HADHA and β-tubulin. B, Quantification of the results shown in panel A. The data are expressed as the means ± s.e.m. of at least six independent experiments. The conventions are same as those shown in Fig. 3. The asterisks on each bar indicate statistical significance between the group and the normal SH-SY5Y cells (Drp1 (cyto): One-way ANOVA, <i>P</i> = 0.413; Drp1 (mito): one-way ANOVA, <i>P</i> = 0.039, post hoc Games-Howell test, **<i>P</i><0.01; Mff: one-way ANOVA, <i>P</i> = 0.011, post hoc Games-Howell test, *<i>P</i><0.05). C, Cells treated as in A were analyzed by immunofluorescence microscopy using anti-HADHA. D, Percentages of cells with the indicated mitochondrial morphology in C. Data were collected from six independent experiments and represent means ± s.e.m. Scale bar, 10 µm (C).</p

Overexpressed truncated DISC1 promotes oligodendrocyte differentiation.

<p><b>A–E</b> Effect of truncated DISC1 overexpression on CNPase and MBP expression. Oligodendrocyte precursor cells were infected with GFP-Adv or trDISC1-GFP-Adv for 12 hours and induced to differentiate by PDGF deprivation for 36 hours. mRNA levels of CNPase (<b>A</b>) and MBP (<b>B</b>) were quantified by qRT-PCR. Data are expressed as mean ± s.e.m. of at least three independent experiments. *<i>p</i><0.05 vs. GFP-Adv. <b>C,</b> Cells infected with GFP-Adv or trDISC1-GFP-Adv for 12 hours were lysed 60 hours after PDGF deprivation and subjected to western blot analysis. <b>D, E,</b> Quantitation of relative band densities for CNPase (<b>D</b>) and MBP (<b>E</b>) was performed by scanning densitometry. Data are expressed as mean ± s.e.m. of at least three independent experiments. *<i>p</i><0.05 vs. GFP-Adv. <b>F–I</b> Oligodendrocyte precursor cells were infected with GFP-Adv or trDISC1-GFP-Adv for 12 hours and induced to differentiate by depriving PDGF for 60 hours then fixed for immunostaining. Cells were immunostained for GFP and β-tubulin (<b>F, G</b>) or for GFP and CNPase (<b>H, I</b>) and analyzed as described in figure legend 3. *<i>p</i><0.05 vs. GFP-Adv. Scale bar = 50 µm.</p