Quantitative PCR analysis of mitochondrial fission/fusion actors and relevant differentially-expressed miRNA-mRNA complexes in human T98G glioblastoma cells.

<p>5A: Quantitative PCR analysis of mitochondrial fission/fusion actors (FIS1 and MFN2) in T98G cells. The data are expressed in units (mRNA expression of a specific gene normalized to β-globin mRNA expression) that are relative to the control, which was assigned a unit value. 5B: Quantitative PCR analysis of relevant differentially expressed miRNA in T98G cells. The data are expressed in units (miRNA expression relative to U5 snRNA) that are relative to the control, which was assigned a unit value. 5C: Quantitative PCR analysis of PTEN and NAIP mRNA, directly targeted by miR-21 and miR-221, respectively. FGFR3 expression was used as negative control of miR-100, which expression level was unchanged by peptide treatment. The data are expressed in units (mRNA expression of a specific gene normalized to β-globin mRNA expression) that are relative to the control, which was assigned a unit value. The values are the average ± SD for three separate determinations (<i>N</i> = 3). *: <i>P</i><0.05 versus control.</p

Action of the NFL-TBS.40-63 peptide on mitochondrial number and functions in T98G cells and NIH 3T3 control cells.

<p>1A: The relative oxygen consumption was measured using a mitostress kit and a Seahorse XF-24 apparatus (from Seahorse Bioscience, North Billerica, MA, USA). The oligomycin-insensitive fraction represents non-phosphorylating respiration, which was recorded after the inhibition of ATP synthase with oligomycin. The oligomycin-sensitive fraction represents the phosphorylating respiration, i.e., the fraction used for ATP synthesis. Results are expressed relative to oxygen consumption of scramble treated cells used as control (pmol/min/mg protein). 1B: The protein expression of mitochondrial subunit IV of complex IV (COX4, MS408, Mitosciences) and subunit Ip of complex II (SDHB, MS203, Mitosciences) were measured by Western blot analysis after a 6-hour exposure to 10 µM NFL-TBS.40-63 peptide and normalized to the α-tubulin level (65 KDa; Abcam, Cambridge, UK). The protein expression for the peptide-treated samples was expressed relative to that of the scramble-treated samples. S: Scramble; P: NFL-TBS.40-63 peptide. Results are expressed relative to protein expression ratio of scramble treated cells used as control. The values represent the average ± SD for three separate determinations (<i>N</i> = 3). *: <i>P</i><0.05 versus control.</p

Effects of 10 µM of NFL-TBS.40-63 peptide on mitochondrial and microtubule networks in human T98G glioblastoma cells.

<p>3A: NFL peptide accumulates within the cell in a polarized manner, limiting the density of the microtubules and mitochondrial networks. 3B: NFL peptide accumulates at the basis of the midbody and excludes the microtubule network. 3C: The peptide surrounds the microtubules' tips and limits filopodia formation. Microtubules were detected using an Alexa647-labeled, anti-α-tubulin antibody (purple); biotinylated NFL-TBS.40-63 peptide was labeled with streptavidin Alexa488 (green), the nuclei with diamidino phénylindole (DAPI; blue) and mitochondria with a mitotracker (RedCMX ROS). The cells were examined with a Nikon A1RSI confocal microscope and the images were analyzed with Nikon NIS-element software. The <i>red bars</i> are the measuring scale.</p

The NFL-TBS.40-63 peptide reorganizes mitochondrial networks in human T98G glioblastoma cells.

<p>The white arrow indicates a mitochondrial network (bottom left) superposed with a microtubule network (bottom right). The yellow arrow indicates a mitochondrial network (bottom left) superposed with long peptide sequences (top right). The microtubules were detected using an Alexa647-labeled, anti-α-tubulin antibody (purple); biotinylated NFL-TBS.40-63 peptide was labeled with streptavidin Alexa488 (green), the nuclei with diamidino ph?nylindole (DAPI; blue) and the mitochondria with a mitotracker Red CMX ROS). The cells were examined with a Nikon A1RSI confocal microscope and the images were analyzed with Nikon NIS-element software. The red bars are the measuring scale.</p

Expression analysis of the genes involved in the control of mitochondrial biogenesis in T98G cells treated by 10 µM of NFL-TBS.40-63 peptide.

<p>2A: Quantitative PCR analysis: PRC (PPRC1) and PGC-1α (PPARGC1A) coactivators, NRF-1 transcription factor, mitochondrial transcription factor TFAM and a component of the respiratory chain, Cytochrome c (CYCS), were measured. The data are expressed in relative units (mRNA expression of a specific gene normalized to β-globin mRNA expression) and expressed relative to the control, which was assigned a unit value. The values are the average ± SD for three separate determinations (<i>N</i> = 3). *: <i>P</i><0.05 versus control. 2B: Western blot analysis: The protein expression of PGC-1α (105KDa, Ab- 54481, Abcam) and NRF-1 (54 KDa, Ab-86516, Abcam) were measured by Western blot analysis after a 6-hours exposure to 10 µM NFL-TBS.40-63 peptide and normalized to the β-actin level (45 KDa; Abcam). Results are expressed relative to protein expression ratio of scramble treated cells which was assigned a unit value. The values are the average ± SD (<i>N</i> = 3). *: <i>P</i><0.05 versus control.</p

ERRα modulates expression and activity of LDH. <i>LDHA</i> and <i>LDHB</i> expression levels were measured by quantitative real-time PCR.

<p>The ratio of LDH activity to CS activity was determined under various conditions. Measurements were made 48 h after transfection or 10 days of treatment with XCT790 and results are presented relative to the control which was assigned a unit value. <i>LDHA</i> and <i>LDHB</i> expression levels, the mean <i>LDHA/LDHB</i> ratio (A) and the relative LDH activity (B) for RO82W-1 cells transfected with 50 ng ERRα or 50 ng ERRα and 50 ng PRC or empty vectors (Control). <i>LDHA, LDHB</i> expression levels and mean of the <i>LDHA/LDHB</i> ratio (C) and relative LDH activity (D) for FTC-133 cells treated with XCT790 or vehicle (Control). <i>LDHA, LDHB</i> expression levels and mean of the <i>LDHA/LDHB</i> ratio (E) and relative LDH activity (F) for FTC-133 cells transfected with control or ERRα siRNA. The results are the mean values±SD of three experiments performed in duplicate relative to controls. *: p≤0.05.</p

Potential ERRα response elements in <i>LDHB</i> and <i>LDHA</i> promoters (A) Potential ERRα binding sites numbered relative to the transcription starting site (TSS) (B) Chromatin ImmunoPrecipitation (ChIP) assay for <i>LDH</i> promoters in XTC.UC1 cells using a polyclonal ERRα antibody.

<p>Chromatin was immunoprecipitated with the indicated antibody and submitted to quantitative PCR. Results are expressed as fold change of enrichment compared to control IgG immunoprecipitated material. ERRα-IP was realized in duplicate and each sample was tested in triplicate for quantitative PCR. TFBS: transcription factor binding site.</p

ERRα and PRC coregulate the cell cycle and metabolism in thyroid cells.

<p>RO82W-1 cells transfected with ERRα and PRC or empty vector (Control) and analysed 72 h after transfection. Pangenomic microarray results for metabolism genes. Gene-expression levels are grouped by class and ordered according to their mean log level of expression (A) or color-coded in the matrix from green (underexpression) to red (overexpression) (B). Ten main gene ontologies (C). Cell proliferation and lactate levels in media (D).</p

Genomic profile.

<p>(A) CGH profile obtained from a pulmonary metastasis of the initial case. Genomic alterations are presented and organized on the X axis from chromosome 1 to 22 and X, Y. Log2 ratio values are reported on the Y axis. Significant gains or losses are indicated by blue lines and blue areas above or below each profile, respectively. Chromosome 2 is highlighted with a black box. (B) Enlargements of chromosome 2 and <i>ALK</i> chromosomal region. The chromosome 2 region containing <i>ALK</i> locus is highlighted with a black box. The log2 ratio values of probes covering the proximal regions of <i>ALK</i> and the gene itself are presented. The arrow indicates the breakage region. (C) Enlargements of chromosome 2 and <i>STRN</i> chromosomal region. The chromosome 2 region containing <i>STRN</i> locus is highlighted with a black box. The log2 ratio values of probes covering the proximal regions of <i>STRN</i> and the gene itself are presented. The arrow indicates the breakage region. (D) Schematic representation of potential breakpoints into the two genes at the genomic level (gDNA) according to the CGH data. Agilent CGH probes surrounding the different breakpoints are indicated.</p

<i>ALK</i>, <i>STRN</i> and <i>STRN/ALK</i> fusion status at mRNA and genomic DNA levels in positive PTC.

<p>(A) Expression profiles of <i>ALK</i>, <i>STRN</i> and <i>STRN/ALK</i> fusion transcript obtained by RT-PCR in two PTC samples and in one control sample (C1) are presented. L: molecular weight ladder, bp: base-pair. (B) Chromatogram showing the sequence of <i>STRN/ALK</i> fusion transcript at the breakpoint observed in the two PTC samples. The <i>STRN</i> exon 3 (NM_003162) at the 5′ part of the transcript is fused to the <i>ALK</i> exon 20 (NM_004304) at the 3′ portion. (C) Products obtained after PCR on genomic DNA using first <i>ALK</i> and <i>STRN</i> forward and reverse primers and then the combination of a <i>STRN</i> forward primer with an <i>ALK</i> reverse primer for case 5 and one control sample (C1). (D) Chromatogram showing the sequence of <i>STRN/ALK</i> fusion at the genomic breakpoint observed in the case 5. One nucleotide at the intronic junction (C) is identical in both fused genes and might be contributed by either of them.</p