Immunocytochemistry and co-immunoprecipitation of UCP4 and mitochondrial Complex II in UCP4-overexpressing SH-SY5Y cells.

<p>(a) UCP4 (red) and a 70 kDa Complex II subunit, Fp (green), were co-localized in mitochondria. (b) Co-immunoprecipitation and western blots of recombinant UCP4 protein and Complex II subunit, Fp. Mitochondrial lysates from UCP4-overexpressing cells were immunoadsorbed (IP) with either anti-FLAG or anti-70 kDa Fp, antibody separately. The resultant antibody-protein pull-down lysates were further adsorbed by Protein-G-sepharose beads. The adsorbed proteins were cross-detected using anti-FLAG and anti-70 kDa Fp in western blots. The pull-down lysate from anti-FLAG showed a band at 70 kDa detected by anti-70 kDa Fp; and the pull-down lysate from anti-70 kDa Fp showed a band at 34 kDa detected by anti-FLAG. IgG-heavy chain and IgG-light chain were detected at ∼55 kDa and ∼25 kDa, respectively. Dashed box showed non-specific bands from crude mitochondrial lysate detected by anti-FLAG.</p

Bioenergetic characterization of SH-SY5Y cells stably overexpressing UCP4.

<p>(a) Human UCP4 protein expression level was significantly higher in UCP4-overexpressing cells than vector controls. (b) Total intracellular ATP level in UCP4-overexpressing cells was higher than vector control cells, as determined by luciferase bioluminescent assay in total cell lysates. (c) Rate of respiration in vector control and UCP4-overexpressing cells. Under normal culture condition, oxygen consumption rate of UCP4-overexpressing cells was higher than vector controls. Results are expressed as mean ± SEM based on at least three independent trials. ** represents statistical significance at p<0.01, * represents p<0.05, compared to the vector control cells.</p

Mitochondrial oxygen consumption and ADP∶oxygen (ADP∶O) ratio of isolated mitochondria from vector control and UCP4-overexpressing cells.

<p>ADP-stimulated (<i>state 3</i>) and resting (<i>state 4</i>) respiration rates are shown for mitochondria maintained in normoxia at 25°C utilizing either (a–b) Complex I or (c–d) Complex II substrates (i.e. glutamate & malate or succinate, respectively). UCP4-overexpressing mitochondria demonstrated similar ADP∶O ratio utilizing (b) Complex I substrates, but was significantly higher when utilizing (d) Complex II substrates, as compared to vector control mitochondria. Results are expressed as mean ± SEM based on at least six independent measurements. * represents statistical significance at p<0.05, compared to the vector control. NS: not significant.</p

Complex II-specific oxygen consumption and ATP production of isolated mitochondria from UCP4-overexpressing and vector control cells.

<p>(a) Oxygraph showing specificity of Complex II-mediated respiration in isolated mitochondria induced by succinate. Addition of rotenone (10 µM) completely blocked Complex I activity, because addition of Complex I substrates (glutamate & malate) and ADP did not induce oxygen consumption. Subsequently, addition of Complex II substrate, succinate, induced oxygen consumption by <i>state 3</i> respiration. (b) Amount of ATP produced in isolated mitochondria from vector and UCP4-overexpressing cells utilizing either Complex I or Complex II substrates. UCP4 overexpression facilitates ATP production using Complex II substrate (succinate). There was no difference in amount of ATP produced when using Complex I substrate (glutamate/malate). Basal value of ATP produced in vector control: glutamate/malate (Complex I) = <i>11.42 nmol ATP/mg mitochondria protein/nmol O₂</i>; succinate (Complex II): <i>6.68 nmol ATP/mg mitochondria protein/nmol O₂</i>. Results are expressed as mean ± SEM based on at least four independent trials. ** represents statistical significance at p<0.01, compared to the vector control cells. NS: not significant.</p

Mitochondrial Complex I, II and IV activity and expression levels in vector control and UCP4-overexpressing cells.

<p>Enzymatic activities of Complex I and II were measured in total cell lysates using spectrophotometric enzyme assay as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032810#s4" target="_blank"><i>Materials and Methods</i></a>. (a)(i–ii) Complex I activity and (c) Complex I subunit, NADH dehydrogenase (ubiquinone) Fe-S protein 4 (NDUFS4) expression levels were similar in both vector control and UCP4-overexpressing cells. (b)(i–ii) Complex II in sample lysates was reactivated in 10 min pre-incubation period before assay. Complex II activity was significantly higher in UCP4-overexpressing cells, but not (c) the Complex-II Fp subunit expression level. (d) Complex IV activity and (c) Complex IV subunit II expression levels were similar in both control and UCP4-overexpressing cells. The expression level of mitochondria marker, CoxIV, reflecting mitochondrial mass, was also similar between UCP4-overexpressing and vector control cells. Results are expressed as mean ± SEM based on at least four independent trials. * represents statistical significance at p<0.05, compared to the vector control cells. NS: Not significant.</p

Level of proton leak and integrity of isolated mitochondria from UCP4-overexpressing and vector control cells.

<p>(a) Oxygraphs showing changes in dissolved oxygen in isolated mitochondria suspension from these cells under substrates-induced respiration. Maximum rate of oxygen consumption was recorded for 2 min after addition of substrates containing 5 mM glutamate & malate, 5 mM succinate, and 0.5 mM ADP. ATP synthase inhibitor, oligomycin (2.5 µg/ml) was then added to block ATP synthesis by Complex V. The rate of oxygen consumption with oligomycin was recorded for an additional 2 min, indicating Complex V-insensitive proton leak. Numbers in brackets represent the rate of oxygen consumption in µmol O₂/min/mg mitochondrial protein. (b) The level of proton leak was defined as the ratio of the rate of oxygen consumption in the presence of oligomycin to the rate of substrates-stimulated oxygen consumption (<i>state 3</i> respiration). Results are expressed as mean ratios ± SEM based on at least three independent trials. ** represents statistical significance at p<0.01, compared to the vector control. (c) Representative oxygraphs reflecting integrity of the outer mitochondrial membrane of isolated mitochondria isolated from both vector control and UCP4-overexpressing cells. Exogenous cytochrome c (Cyt <i>c</i>) did not further increase mitochondrial oxygen consumption. Intact and functional mitochondria were demonstrated by <i>state 3</i> respiration as stimulated by addition of substrates (glutamate/malate/succinate) and ADP.</p

Classical <i>state 4</i> and <i>state 3</i> respiration rate in UCP4-overexpressing and control mitochondria.

<p>Classical <i>state 4</i> and <i>state 3</i> respiration rate in UCP4-overexpressing and control mitochondria.</p

<i>Hsa-miR-765</i> suppresses DU145 cell growth, migration, and invasion.

<p>(A) <i>Hsa-miR-765</i> mimic effectively recognizes reporter with complementary sequence of <i>hsa-miR-765</i> in DU145 cells. Fold changes of luciferase activities of the <i>hsa-miR-765</i> mimic treated cells relative to the cells treated with the negative-control mimic are presented (n = 3). Transfection reagents were used as control. (B) <i>Hsa-miR-765</i> mimic reduces DU145 cell growth. MTS assay was performed on the cells treated with <i>hsa-miR-765</i> mimic or negative-control mimic or transfection control for 4 days (n = 8). (C) <i>Hsa-miR-765</i> mimic significant reduces G0/G1 to G2/M ratio in DU145. Representative DNA histograms (n = 3) are presented. (D) <i>Hsa-miR-765</i> mimic treatment causes up-regulation of cyclin A, cyclin B, and phosphorylated-cdc2 expression in DU145 cells. Protein expression levels of cell cycle regulator proteins were determined by Western blot analyses. Two independent experiments were performed and one representative set of data was presented. (E) <i>Hsa-miR-765</i> mimic suppresses DU145 cell migration and invasion as shown in transwell migration assay (top left) and invasion assay (top right), respectively. Representative micrographs of the cells after transwell migration (top left) or invasion assay (top right) are presented. Fold changes of migration (bottom left) and invasion (bottom right) of DU145 cells with either <i>hsa-miR-765</i> mimic or negative-control mimic relative to the control cells with negative-control mimic are presented (n = 3). (F) <i>Hsa-miR-765</i> mimic significantly reduces stress fibers and filopodia formations in DU145 cells. Representative micrographs and the percentages of the cells with intense stress fibers and the filopodial cells (n = 3) are presented. Student's t-test was used for comparisons with a cutoff p value of 0.05. ** p<0.01; bar = S.D.</p

HMGA1 is a direct target of <i>hsa-miR-765</i>.

<p>(A) The 3′UTR of <i>HMGA1</i> from +8910 to +8929 is predicted to be <i>hsa-miR-765</i> binding site. (B) <i>Hsa-miR-765</i> interacts with 3′UTR of <i>HMGA1</i> in a targeting reporter assay. DU145 cells were transfected with either pMIR-empty or pMIR-HMGA1-3UTR in which 3′ UTR of <i>HMGA1</i> (+8026–+9332) was cloned into the 3′ end of luciferase. Reporter activities of the pMIR-HMGA1-3UTR transfected cells treated with <i>hsa-miR-765</i> mimic or negative-control mimic are compared (n = 3). (C) <i>Hsa-miR-765</i> mimic reduced HMGA1 protein expression in DU145 cells. Protein and mRNA levels of HMGA1 in the <i>hsa-miR-765</i> mimic- and negative-control mimic-treated cells were determined by Western blot analysis (upper) and real-time RT-PCR analysis (lower), respectively. Results from <i>miR-765</i> mimic vs negative control mimic are compared (n = 3). (D) Fulvestrant reduces HMGA1 protein expression in DU145 cells. Protein level of HMGA1 and β-actin in the fulvestrant-treated and ethanol-treated control (CTL) cells were determined by Western blot analysis. (E) Ectopic expression of HMGA1 blocks fulvestrant-induced DU145 cell growth inhibition. The relative cell growth was determined after 4 days of treatment with fulvestrant or ethanol after stable transfection of <i>HMGA1</i> (or empty vector for control) for a week. Protein levels of HMGA1 were shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098037#pone.0098037.s006" target="_blank">Figure S6</a>. The cell growth of fulvestrant-treated cells with HMGA1 overexpression vs empty vector are compared (n = 8). Student's t-test was performed to determine significance between groups using a cutoff p value of 0.05. **p<0.01; bar = S.D.</p

Fulvestrant inhibits DU145 cell growth, migration, and invasion.

<p>(A) Fulvestrant induces growth inhibition of DU145 cells via an ERβ-dependent mechanism. Growth of the fulvestrant-treated DU145 cells with or without ERβ siRNA knockdown for 4 days relative to the ethanol-treated control cells with negative-control siRNA are presented and compared (n = 8). ERβ expression was also knocked down by another siRNA (siRNA#2) and the similar results were obtained (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098037#pone.0098037.s005" target="_blank">Figure S5</a>). (B) Fulvestrant induces DU145 cell-cycle arrest at G2/M phase. Representative DNA histograms of 48 hrs fulvestrant -or ethanol- (control) treated cells and percentage distributions of the cells at G0/G1 and G2/M phases (n = 3) are presented and compared. (C) Fulvestrant induces expression of G2/M markers. DU145 cells were treated with fulvestrant or ethanol for 2 days (control) and cell cycle markers were determined by Western blot analysis. Two independent experiments were performed and one representative set of data was presented. (D) Fulvestrant suppresses cell migration. A wound-healing assay was performed on the fulvestrant- and ethanol (EtOH)-treated DU145 cells (n = 3). Representative micrographs of the fulvestrant- and ethanol-treated cell cultures with scratches at 0 h and after 16 h are shown. The wound is marked by dotted lines. (E) Fulvestrant inhibits transwell migration (left panel) and invasion (right panel) in DU145 cells (n = 3) after 5 hrs of fulvestrant treatment. (F) Reductions of filopodial cells and cells with intense stress fibers by fulvestrant (treated with 48 hrs) via an ERβ-dependent mechanism. Representative micrographs and the percentages of the cells with intense stress fibers and the filopodial cells (n = 3) are presented. Student t-test was performed to determine significance with a cutoff p value of 0.05. ** p<0.01; bars = S.D.</p