TNF-α induced mtDNA depletion, lesions and repair.

<p>(A) Cells were pretreated or not (TNF-α) for 1 h with 1 µg/ml TNF-R1 antibody (TNF-R1 Ab) or with 5 mM NAC. They were then treated for 0 to 6 h with 30 ng/ml TNF-α. To evaluate mtDNA depletion, total genomic DNA was isolated and quantification of mtDNA performed by simultaneous real-time qPCR amplification of fragments encoding mitochondrial 12S rRNA and nuclear 18S rRNA used as a reference gene. Results are expressed in 12S mtDNA over 18S nDNA relative ratio (mean values ± SEM of four independent experiments with four replicates, *p<0.05). (B) mtDNA lesions per 10 Kb were quantified by qPCR amplification of a large fragment (8.9 Kb) from cells treated or not for 15 min-6 h with 30 ng/ml TNF-α and expressed using the Poisson expression <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040879#pone.0040879-Santos1" target="_blank">[27]</a> (mean values ± SEM of three independent experiments with three replicates, *p<0.05). (C) mtDNA repair activity was measured by calculating relative amplification comparing the values of the treated samples with undamaged control <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040879#pone.0040879-Santos1" target="_blank">[27]</a>, a 50% mtDNA control has been performed (mean values ± SEM of three independent experiments with three replicates, *p<0.05).</p

TNF-α induced p53 translocation to mitochondria.

<p>(A) Accumulation of p53 was investigated by Western Blot using p53 antibody in lysate from cells treated for 1 h with 5–100 ng/ml TNF-α or with 30 ng/ml TNF-α for 0 to 180 min. β-actin served as a loading control (B) Accumulation of phosphoSer¹⁵p53 was investigated by Western Blot in lysate from cells treated for 0–180 min with 30 ng/ml TNF-α. β-actin served as a loading control. (C) Mitochondrial (mito) and cytoplasmic fractions (cyto) were isolated as described in Material and Methods and their purities checked by Western Blot using β-actin antibody (cytoplasm) and COXI (mitochondria). (D) The translocation of p53 to mitochondria was investigated by Western Blot using p53 antibody on mitochondrial (mito) or cytoplasmic (cyto) fractions isolated from cells treated or not for 0 to 60 min with 30 ng/ml TNF-α. COXI and β-actin served as loading controls. (E) Cells were pretreated or not (control) for 1 h with 5 mM NAC before exposure to TNF-α. Western Blots were performed using p53 antibody. COXI was used as a loading control.</p

TNF-α induced ROS, 8-oxo-dG production and mtDNA repair.

<p>(A) TNF-α induced extra and intracellular ROS were measured over 1 h-period using LAC assay on cell suspension (10⁶ cells in 0.5 ml Hanks buffer) as described in Materials and Methods. One representative experiment of four independent studies is shown. (B) Chemiluminescence is also quantified at the peak in the absence or presence of 5 mM NAC as a percentage of basal value (control) (mean ± SEM for three independent experiments, *p<0.05). (C) TNF-α induced levels of 8-oxo-dG after 15 min to 3 h cell treatment were measured using the OxiSelect™ Oxidative DNA damage ELISA kit (mean values ± SEM of three independent experiments *p<0.05). (D) The decrease of mtDNA remaining ARP-reactive sites created at 30 min TNF-α treatment (100%) was measured from 1 to 6 h using the OxiSelect™ oxidative DNA damage quantitation kit (mean values ± SEM of three independent experiments *p<0.05 <i>vs</i> 30 min).</p

SB216763 or GSK3β siRNAs inhibited mtDNA depletion whereas GSK3βS9A suppressed the reversion.

<p>(A) All siRNA transfections were performed or not (control, C) with 12.5 nM siRNAs directed against GSK3β mRNA (GSK3β siRNAs) or non-targeting (NT) siRNAs in DharmaFECT4® transfection reagent. To check SiRNA transfection efficiency, Western Blots were performed at 48 h with GSK3β or GAPDH (loading control) antibody. (B) Inhibition of GSK3β expression by siRNAs relative to GAPDH was quantified using the Bio1D software (mean values ± SEM of three experiments *p<0.05) (C) Cells were transfected or not (control C or Fugene HD® alone, F) with the mutant GSK3βS9A pcDNA3 plasmid or the empty plasmid (pcDNA3) using Fugene HD® (F). After 72 h transfection, the expression of recombinant GSK3βS9A protein was checked by Western Blot using GSK3β or GAPDH (loading control) antibody. (D) Cells were treated or not with SB216763 or transfected or not (untransfected cells) with NT siRNAs or GSK3β siRNAs or GSK3βS9A pcDNA3 plasmid. Then, they were treated for 0 (zero-time control) to 6 h with 30 ng/ml TNF-α. Total DNA was isolated and the quantification of mtDNA content performed by real-time qPCR co-amplification of fragments encoding mitochondrial 12S rRNA and nuclear 18S rRNA as a gene reference. Results are expressed in mtDNA over nDNA relative ratio (mean values ± SEM of three independent experiments with five replicates, *p<0.05).</p

TNF-α did not induce apoptosis of HepG2 cells.

<p>(A) Western Blot using the TNF-R1 receptor antibody was performed on cell lysate. (B) PARP cleavage was investigated by Western Blot in cells treated for 18 h with 30 or 100 ng/ml TNF-α or with 1 µM doxorubicin (Doxo) as a positive control. (C) The lack of apoptotic bodies in basal or cells treated with 30 or 100 ng/ml TNF-α has been confirmed by DAPI staining and UV-microscopy.</p

p53 inhibition by siRNAs and phosphoSer¹⁵p53 antibody prevented the reversion of mtDNA depletion.

<p>(A) All siRNA transfections were performed with 12.5 nM siRNAs directed against p53 mRNA (p53 siRNAs) or non-targeting siRNAs (NT) and DharmaFECT4® transfection reagent used as a control (c). To check siRNA transfection efficiency, Western Blots were performed at 48 h using p53 or GAPDH (loading control) antibody. (B) The knockdown of p53 expression by siRNAs relative to GAPDH was quantified using the Bio1D software (mean values ± SEM of three experiments *p<0.05) (C) DNA was isolated from untransfected cells or transfected with NT siRNAs or with p53 siRNAs. Cells were also permeabilized with 0.1% Triton X100 and pretreated for 1 h with 1 µg/ml phosphoSer¹⁵p53 antibody (phosphoSer¹⁵Ab). Cells were then treated for 0 to 6 h with 30 ng/ml TNF-α. The quantification of mtDNA content was performed by simultaneous real-time qPCR amplification of fragments encoding mitochondrial 12S rRNA and nuclear 18S rRNA serving as a reference gene. Results are expressed in mtDNA over nDNA relative ratio (mean values ± SEM of three independent experiments with five replicates, *p<0.05).</p

TNF-α induced p53 interaction to GSK3β, TFAM and D-loop.

<p>Cells were treated for 0 (zero-time control) or 1 h with 30 ng/ml TNF-α. Mitochondrial fractions were isolated and co-immunoprecipitated or not (input) with GSK3β, p53 (FL-393) or TFAM polyclonal antibodies, IgG or no antibody was used as a control (c). (A) Western Blots were performed using GSK3β or p53 (DO1) antibody. (B) Western Blots were performed using phosphoSer¹⁵p53 or phosphoSer⁹GSK3β antibody in not pretreated or pretreated cells with GSK3β inhibitor SB216763. (C) Western Blots were performed using TFAM or GSK3β antibody. (D) The mtDIP assay was performed with cross-linked DNA prepared from treated cells for 0 (zero-time control) or 1 h with 30 ng/ml TNF-α. Immunoprecipitates were performed without (control, c) or with IgG used as a control or with p53 antibody. PCR were realized on immunoprecipitates or inputs using a primer pair covering D-loop, cytochrome b (cyt b) or ATPase 6 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040879#pone-0040879-g005" target="_blank">Figure 5D</a>).</p

A Novel Lamin A Mutant Responsible for Congenital Muscular Dystrophy Causes Distinct Abnormalities of the Cell Nucleus

<div><p>A-type lamins, the intermediate filament proteins participating in nuclear structure and function, are encoded by <i>LMNA</i>. <i>LMNA</i> mutations can lead to laminopathies such as lipodystrophies, premature aging syndromes (progeria) and muscular dystrophies. Here, we identified a novel heterozygous <i>LMNA</i> p.R388P de novo mutation in a patient with a non-previously described severe phenotype comprising congenital muscular dystrophy (L-CMD) and lipodystrophy. In culture, the patient’s skin fibroblasts entered prematurely into senescence, and some nuclei showed a lamina honeycomb pattern. C2C12 myoblasts were transfected with a construct carrying the patient’s mutation; R388P-lamin A (LA) predominantly accumulated within the nucleoplasm and was depleted at the nuclear periphery, altering the anchorage of the inner nuclear membrane protein emerin and the nucleoplasmic protein LAP2-alpha. The mutant LA triggered a frequent and severe nuclear dysmorphy that occurred independently of prelamin A processing, as well as increased histone H3K9 acetylation. Nuclear dysmorphy was not significantly improved when transfected cells were treated with drugs disrupting microtubules or actin filaments or modifying the global histone acetylation pattern. Therefore, releasing any force exerted at the nuclear envelope by the cytoskeleton or chromatin did not rescue nuclear shape, in contrast to what was previously shown in Hutchinson-Gilford progeria due to other <i>LMNA</i> mutations. Our results point to the specific cytotoxic effect of the R388P-lamin A mutant, which is clinically related to a rare and severe multisystemic laminopathy phenotype.</p></div

Abnormal cellular phenotypes of fibroblasts from the patient with <i>LMNA</i> p.R388P-associated L-CMD and lipodystrophy.

<p><b>A)</b> Phase-contrast images of control (passage 11) and patient (passage 9) cells. Scale bar, 100 μm. <b>B)</b> Measurement of nuclear area for control and patient fibroblasts at passages 13 and 11, respectively (mean ± s.e.m.). n > 100 nuclei/condition. <b>C)</b> Senescence assessment using the β-Galactosidase assay for control and patient fibroblasts at passages 9 and 6, respectively. <b>D)</b> Whole cell extracts of fibroblasts from control and patient at passages 10 and 14 (P10, P14) were analysed by western blot using antibodies against LA/C, LB1 or GAPDH. ECL signals were then scanned and quantified. The graph illustrates LA and LC ECL signal intensities as relative optical densities (O.D.) normalised versus GAPDH (n = 4) (mean ± s.e.m.). <b>E)</b> Human skin fibroblasts from control (passage 13) and patient (passage 11) were fixed, labelled with anti-LA/C or anti-LB1 antibodies, and observed by immunofluorescence microscopy. DNA was stained with Hoechst. Shown are images representative for normal nuclear morphology (Normal), nuclear dysmorphy (Dysm.) and lamina with a honeycomb pattern (Hon.). Asterisks indicate the honeycomb aspect of the lamin A/C network. Arrows indicate the regions with weak lamin B1 staining. The percentages of the different phenotypes are indicated on the pictures; n > 300 nuclei/condition. Scale bar, 10 μm.</p

Changes in the lamin A in situ proximity with LAP2α and emerin in response to R388P-LA expression.

<p><b>A,E)</b> Whole cell extracts of C2C12 cells either control (Ctrl) or overexpressing wild-type (WT) or R388P (RP) LA, tagged with GFP (left panel) or FLAG (right panel) were analysed by western blot using anti-GFP, anti-FLAG, anti-LAP2α, anti-emerin and anti-GAPDH antibodies, as indicated. <b>B-D, F-H)</b> C2C12 cells overexpressing WT (upper panel) or R388P (lower panels) GFP-LA or FLAG-LA were fixed, labelled with anti-GFP and anti-LAP2α or anti-FLAG and anti-emerin antibodies, and processed either for immunofluorescence <b>(B,F)</b> or proximity ligation assay (PLAssay) <b>(C,G)</b>, before observation at the confocal microscope. Scale bar, 10 μm. <b>D,H)</b> Quantification of PLA signals per nucleus among GFP/PLA or FLAG/LA positive cells as shown in <b>C,G)</b>. The graphs show the median intensity of the total PLA signals detected per nucleus (upper panel) and the median frequency of the PLA signals detected at the intranuclear periphery (lower panel). Boxes show first and third quartiles, bars are put according to Tukey method for n = 59 (WT) and 31 (R388P) nuclei for <b>D</b> and n = 67 (WT) and 74 (R388P) nuclei for <b>H</b> *** p < 0.001 (Mann Whitney test).</p