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• Editorial Volume 13, Issue 11 pp 14546-14548

  NAD⁺ deficit, protein acetylation and muscle aging

  Relevance score: 7.325934

  Li Li Ji, Dongwook Yeo

  Keywords: aging, SIRT, NAD ⁺, deacetylation, skeletal muscle

  Published in Aging on June 7, 2021

• Review Volume 10, Issue 8 pp 1801-1824

  Liver regeneration in aged mice: new insights

  Relevance score: 5.5955753

  Monica Pibiri

  Keywords: ageing, 2/3 PH, BubR1, YAP, SIRT-1, HSCs, SASP, autophagy

  Published in Aging on August 28, 2018

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  The regenerative capacity of the liver after resection is reduced with aging. Recent studies on rodents revealed that both intracellular and extracellular factors are involved in the impairment of liver mass recovery during aging. Among the intracellular factors, age-dependent decrease of BubR1 (budding uninhibited by benzimidazole-related 1), YAP (Yes-associated protein) and SIRT1 (Sirtuin-1) have been associated to dampening of tissue reconstitution and inhibition of cell cycle genes following partial hepatectomy. Extra-cellular factors, such as age-dependent changes in hepatic stellate cells affect liver regeneration through inhibition of progenitor cells and reduction of liver perfusion. Furthermore, chronic release of pro-inflammatory proteins by senescent cells (SASP) affects cell proliferation suggesting that senescent cell clearance might improve tissue regeneration. Accordingly, young plasma restores liver regeneration in aged animals through autophagy re-establishment. This review will discuss how intracellular and extracellular factors cooperate to guarantee a proper liver regeneration and the possible causes of its impairment during aging. The possibility that an improvement of the liver regenerative capacity in elderly might be achieved through elimination of senescent cells via autophagy or by administration of direct mitogenic agents devoid of cytotoxicity will also be entertained.

  

  Intracellular factors affecting compensatory regeneration in aged livers.Young livers (left): reduction of the liver mass after PH leads to YAP/Sirtuin-dependent transcription of BubR1. BubR1, in turn, induces the cell adhesion protein DISC1 which provides the proper microstructural adaptation during regeneration. Aged livers (right): the increased levels of MSTs counteract YAP activation. Furthermore, as aging is associated to decreased SIRT-1 expression, there is a reduction of the YAP/Sirtuin-dependent transcription of BubR1. This lead to reduction of DSC1 expression, weakened microstructural adaptation and upregulation of p21. As a result, G1 cell cycle arrest and impairment of liver regeneration is observed.

  
  
  

  

  Extracellular factors affecting compensatory regeneration in aged livers. During ageing, activated HSC-induced chemokine production leads to a decreased activation and proliferation of Liver Progenitor Cells (LPCs). Since these cells are required to repopulate the liver following tissue injury, their decrease results in a reduced liver regenerative response. Furthermore, age-dependent HSC size increase and migration, can negatively affect hepatocyte proliferation by reducing liver perfusion. A further reason for the impaired liver regeneration of aged hepatocytes is due to the pro-inflammatory proteins chronically released by senescent hepatocytes that accumulate in the elderly as a consequence of a reduction of autophagy program.

  
  
  

• Review Volume 9, Issue 6 pp 1477-1536

  Effects of resveratrol, curcumin, berberine and other nutraceuticals on aging, cancer development, cancer stem cells and microRNAs

  Relevance score: 6.9861426

  James A. McCubrey, Kvin Lertpiriyapong, Linda S. Steelman, Steve L. Abrams, Li V. Yang, Ramiro M. Murata, Pedro L. Rosalen, Aurora Scalisi, Luca M. Neri, Lucio Cocco, Stefano Ratti, Alberto M. Martelli, Piotr Laidler, Joanna Dulińska-Litewka, Dariusz Rakus, Agnieszka Gizak, Paolo Lombardi, Ferdinando Nicoletti, Saverio Candido, Massimo Libra, Giuseppe Montalto, Melchiorre Cervello

  Keywords: miRs, SIRT, gene methylation, CSCs, natural products, resveratrol, curcumin

  Published in Aging on June 12, 2017

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  Natural products or nutraceuticals have been shown to elicit anti-aging, anti-cancer and other health-enhancing effects. A key target of the effects of natural products may be the regulation of microRNA (miR) expression which results in cell death or prevents aging, diabetes, cardiovascular and other diseases. This review will focus on a few natural products, especially on resveratrol (RES), curcumin (CUR) and berberine (BBR). RES is obtained from the skins of grapes and other fruits and berries. RES may extend human lifespan by activating the sirtuins and SIRT1 molecules. CUR is isolated from the root of turmeric (Curcuma longa). CUR is currently used in the treatment of many disorders, especially in those involving an inflammatory process. CUR and modified derivatives have been shown to have potent anti-cancer effects, especially on cancer stem cells (CSC). BBR is also isolated from various plants (e.g., Coptis chinensis) and has been used for centuries in traditional medicine to treat diseases such as adult- onset diabetes. Understanding the benefits of these and other nutraceuticals may result in approaches to improve human health.

  

  Pleiotropic effects of resveratrol on signaling pathways involved in cell growth. Some of the pathways affected by RES are indicated. This diagram focusses on signaling pathways predominately involved in aging and cancer. Red arrows indicate induction of an event; black closed arrows indicate suppression of an event.

  
  
  

  

  Pleiotropic effects of curcumin on signaling pathways involved in cell growth. Curcumin can induce many pathways which may result in suppression of cell growth, induction of apoptosis, autophagy, senescence or inhibition of cell cycle progression. These events are indicated by red arrows. In addition, CUR treatment can result in the suppression of many important proteins which result in decreased growth, EMT, metastasis or inflammation. These events are indicated by black closed arrows.

  
  
  

  

  Effects of curcumin on TP53 and apoptosis pathways. An overview of the effects of CUR on TP53 and apoptotic pathways and the effects of miRs are indicated. miRs in magenta font indicate oncomirs. miRs in blue font are tumor suppressor miRs. Red arrows indicate induction of an event; black closed arrows indicate suppression of an event.

  
  
  

  

  Effects of curcumin on chromatin structure. An overview of the effects of CUR on demethylation of genes and the effects on the various genes are indicated. Red arrows indicate induction of an event; black closed arrows indicate suppression of an event. M = methylation of a sequence.

  
  
  

  

  Effects of curcumin on drug sensitivity pathways. An overview of the effects of CUR on drug sensitivity pathways and the effects of miRs are indicated. Red arrows indicate induction of an event; black closed arrows indicate suppression of an event.

  
  
  

  

  Overview of effect of curcumin on stemness, microenvironment and EMT. An overview of the effects of CUR on pathways involving stemness, microenvironment and EMT pathways and the effects of miRs are indicated. Red arrows indicate induction of an event; black closed arrows indicate suppression of an event.

  
  
  

  

  Effects of curcumin on CRC chemoresistance. An overview of the effects of CUR on CRC chemoresistance and the effects of miRs are indicated. Red arrows indicate induction of an event; black closed arrows indicate suppression of an event.

  
  
  

  

  Effects of curcumin on HCC. An overview of the effects of CUR on HCC is presented. M = methylated residue, A = acetlylated residue. Red arrows indicate induction of an event; black closed arrows indicate suppression of an event.

  
  
  

  

  Overview of effects of curcumin on HIF-1alpha, miRs and metastasis. An overview of the effects of CUR on pathways involving migration, invasion and metastatic pathways are indicated. Red arrows indicate induction of an event; black closed arrows indicate suppression of an event.

  
  
  

  

  Pleiotropic effects of berberine on signaling pathways involved in cell growth. BBRs can induce many pathways which may result in suppression of cell growth, induction of apoptosis, autophagy, senescence, DNA double strand breaks or inhibition of cell cycle progression and DNA replication. These events are indicated by red arrows. In addition, BBR treatment can result in the suppression of many important proteins leading to decreased growth, decreased DNA synthesis, mitochondrial membrane potential, cell cycle arrest and inflammation indicated in black closed arrows.

  
  
  

  

  Effects of berberine on TP53, miRs, apoptosis and drug resistance. Berberine can induce the expression of TP53 and miRs which can alter the expression of many genes involved in the regulation of apoptosis, cell cycle progression and drug resistance. miRs which are oncomirs are indicated in maroon, miRs which are tumor suppressor miRs are indicated in blue.

  
  
  

  

  Chemical structures of modified berberines from naxospharma.

  
  
  

• Research Paper Volume 6, Issue 10 pp 820-834

  Dysregulation of SIRT-1 in aging mice increases skeletal muscle fatigue by a PARP-1-dependent mechanism

  Relevance score: 5.8396325

  Junaith S. Mohamed, Joseph C. Wilson, Matthew J. Myers, Kayla J. Sisson, Stephen E. Alway

  Keywords: Aging, oxidative stress, PARP-1, sarcopenia, SIRT-1, skeletal muscle

  Published in Aging on October 28, 2014

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  Accumulation of reactive oxygen species (ROS) in skeletal muscles and the resulting decline in muscle performance are hallmarks of sarcopenia. However, the precise mechanism by which ROS results in a decline in muscle performance is unclear. We demonstrate that isometric-exercise concomitantly increases the activities of Silent information regulator 1 (SIRT-1) and Poly [ADP-ribose] polymerase (PARP-1), and that activated SIRT-1 physically binds with and inhibits PARP-1 activity by a deacetylation dependent mechanism in skeletal muscle from young mice. In contrast, skeletal muscle from aged mice displays higher PARP-1 activity and lower SIRT-1 activity due to decreased intracellular NAD⁺ content, and as a result reduced muscle performance in response to exercise. Interestingly, injection of PJ34, a PARP-1 inhibitor, in aged mice increased SIRT-1 activity by preserving intracellular NAD⁺ content, which resulted in higher skeletal muscle mitochondrial biogenesis and performance. We found that the higher activity of PARP-1 in H₂O₂-treated myotubes or in exercised-skeletal muscles from aged mice is due to an elevated level of PARP-1 acetylation by the histone acetyltransferase General control of amino acid synthesis protein 5-like 2 (GCN-5). These results suggest that activation of SIRT-1 and/or inhibition of PARP-1 may ameliorate skeletal muscle performance in pathophysiological conditions such as sarcopenia and disuse-induced atrophy in aging.

  

  Total RNA and cell lysates were isolated from the control or exercised gastrocnemius muscle. (A) Global cellular protein PARylation was determined in total cell lysates by immunoblots. PARP-1 (B) and SIRT-1 (C) mRNA (top) and protein (bottom) levels were determined in total muscle mRNA and cell lysate, respectively. GAPDH was used as a loading control. (D) PGC-1α acetylation levels were estimated by immunoblotting after IP. (E) mRNA expression of the indicated genes in the total RNA was examined by qPCR. (F) mtDNA was evaluated in total muscle genomic DNA by qPCR. (G) PARP-1 acetylation levels were estimated by immunoblotting after IP. (H) SIRT-1 and PARP-1 or (I) GCN5 and PARP-1 binding assays were estimated by immunoblotting after IP. (J) GCN5 protein levels were determined in total cell lysates by immunoblots. The blots are representative of three independent experiments. The data are presented as mean ± SEM (n = 3). White and black bars indicate non-exercised and exercised gastrocnemius muscle, respectively. COX17, cyclooxygenase 17; CytC, Cytochrome C; ERR-α, estrogen-related receptor α; mtTFA, mitochondrial transcription factor A; Ndufa2, NADH dehydrogenase [ubiquinone] iron-sulfur protein a 2; NRF1,nuclear respiratory factor 1; MCD, medium-chain acyl-CoA dehydrogenase; MCAD, medium-chain acyl-CoA dehydrogenase; SDH, succinate dehydrogenase; Tropn I, troponin I; UCP2, uncoupling protein 2; immunoprecipitation (IP).

  
  
  

  

  Total RNA and cell lysates were isolated from the control or exercised gastrocnemius muscles. (A) Global cellular protein PARylation was determined in total cell lysate by immunoblots. (B) NAD+ levels in skeletal muscle were determined in control and exercised muscles. PARP-1 (C) and SIRT-1 (D) mRNA (top) and protein (bottom) levels were determined in total muscle mRNA and cell lysates, respectively. GAPDH was used as a loading control. (E) PGC-1α acetylation levels were estimated by immunoblotting after IP. (F) SIRT-1 and PARP-1 binding assays, PARP-1 acetylation levels (G) or PARP-1 and GCN5 binding assays (H) were estimated by immunoblotting after IP. (I) GCN5 mRNA (top) and protein (bottom) levels were determined in total muscle mRNA and cell lysate, respectively. (J) mRNA expression of the indicated genes in the total RNA was examined by qPCR. (K) Maximal evoked isometric forces from 350 contractions are shown for the plantar flexor muscles in young and aged mice. All force measurements were normalized to body weight (g). The blots are representative of three independent experiments. The data are presented as mean ± SEM (n = 3). White and black bars indicate non-exercised and exercised gastrocnemius muscles, respectively. COX17, cyclooxygenase 17; CytC, Cytochrome C; ERR-α, estrogen-related receptor α; mtTFA, mitochondrial transcription factor A; Ndufa2, NADH dehydrogenase [ubiquinone] iron-sulfur protein a 2; NRF1,nuclear respiratory factor 1; MCD, medium-chain acyl-CoA dehydrogenase; MCAD, medium-chain acyl-CoA dehydrogenase; SDH, succinate dehydrogenase; Tropn I, troponin I; UCP2, uncoupling protein 2; immunoprecipitation (IP).

  
  
  

  

  Total RNA and cell lysates were isolated from the skeletal muscle of mice that were exercised and PBS injected (Ex) or exercised and PJ34 injected (Ex + PJ34). (A) Global cellular protein PARylation was determined in total cell lysates by immunoblots. (B) NAD⁺ levels were determined in in skeletal muscle of Ex or Ex + PJ34 mice. (C) PGC-1α acetylation levels were estimated by immunoblotting after IP. PARP-1 (D) and SIRT-1 (E) mRNA (top) and protein (bottom) levels were determined in total muscle mRNA and cell lysates, respectively. GAPDH was used as a loading control. PARP-1 acetylation levels (F), SIRT-1 and PARP-1 binding assays (G), or PARP-1 and GCN5 binding assays (H) were estimated by immunoblotting after IP. (I) mRNA expression of the indicated genes in the total RNA was examined by qPCR. (J) mtDNA was evaluated in total muscle genomic DNA by qPCR. (K) Maximal isometric forces from the plantar flexor muscles are presented for Ex or Ex + PJ34 mice. All force measurements were normalized to body weight (g). The blots are representative of three independent experiments. The data are presented as mean ± SEM (n = 3). White and black bars indicate Ex and Ex + PJ34 mice, respectively. COX17, cyclooxygenase 17; CytC, Cytochrome C; ERR-α, estrogen-related receptor α; mtTFA, mitochondrial transcription factor A; Ndufa2, NADH dehydrogenase [ubiquinone] iron-sulfur protein a 2; NRF1,nuclear respiratory factor 1; MCD, medium-chain acyl-CoA dehydrogenase; MCAD, medium-chain acyl-CoA dehydrogenase; SDH, succinate dehydrogenase; Tropn I, troponin I; UCP2, uncoupling protein 2. IP, immunoprecipitation.

  
  
  

  

  Total RNA and cell lysates were isolated from myotubes either treated or not treated with H₂O₂. (A) Global cellular protein PARylation was determined in the total cell lysate by immunoblots. (B) PARP-1 mRNA and protein levels were determined in total mRNA and cell lysates, respectively. GAPDH was used as a loading control. (C) Myotube survival (%) in the presence or absence of H₂O₂ was determined by MTT assay at the indicated time-points. (D) Global cellular protein PARylation was determined in total cell lysates from either H₂O₂-treated or non-treated myotubes either in the presence or absence of PJ34 by immunoblots. (E) Myotube survival (%) was determined by the MTT assay in myotubes treated with the conditions similar to ‘D’. (F) Myotubes were transfected with non-specific or PARP-1-targeting siRNAs. PARP-1 protein abundance were analyzed 48 h after transfection by immunoblots. (G) Global cellular protein PARylation was determined in total cell lysates from either H₂O₂-treated or non-treated myotubes either in the presence or absence of PARP-1 by immunoblots. (H) Myotube survival (%) was determined by a MTT assay in myotubes treated with the conditions similar to ‘G’. (I) SIRT-1 mRNA (left side) and protein (right side) levels were determined in total mRNA and cell lysates, respectively. (J) PARP-1 acetylation levels were estimated from immunoblots after IP with the conditions similar to ‘G’. (K) PGC-1α acetylation levels were determined in total cell lysates from either myotubes treated with H₂O₂ or non-treated myotubes with or without PJ34 or RSV by immunoblotting after IP. Global cellular protein PARylation (L) or myotube survival (%) was determined in total cell lysates from either myotubes treated with H₂O₂ or non-treated myotubes with or without resveratrol. IP, immunoprecipitation; RSV, resveratrol.

  
  
  

  

  (A) NAD⁺ levels were determined in myotubes either in the presence or in the absence of H₂O₂ with or without the inhibition of PARP-1 by PJ34 or siRNA. (B) NAD⁺ levels were determined in myotubes either in the presence or in the absence of H₂O₂ with or without the inhibition of PARP-1 by PJ34 or siRNA. (B and C) SIRT-1 protein PARylation was determined in total cell lysates from myotubes treated with the conditions similar to ‘A’ by immunoblotting after IP. (D) PGC-1α acetylation levels were determined by immunoblotting in total cell lysates from myotubes treated with or without resveratrol. (E) SIRT-1 protein PARylation was determined in myotubes either in the presence or in the absence of H₂O₂ with or without RSV. (F) Infection of myotubes isolated from conditional knockout mice (flox/flox) with AAV2-GFP or AAV2-Cre-GFP to determine SIRT-1 knock-down. (G) PGC-1α acetylation levels were determined in total cell lysate from myotubes infected with either AAV2-GFP or AAV2-Cre-GFP in the presence or absence of RSV by immunoblotting after IP. Global cellular protein PARylation (H) and percentage of myotube survival (I) were determined in either AAV2-GFP or AAV2-Cre-GFP infected myotubes in the presence or in the absence of H₂O₂ or RSV. AAV, adeno-associated virus; GFP, green fluorescence protein; IP, immunoprecipitation; RSV, resveratrol.

  
  
  

  

  In young mice, SIRT-1 inhibits the exercise-induced PARP-1 activity by deacetylation-dependent mechanism and as a result increases skeletal muscle performance via enhanced mitochondrial biogenesis. In contrast, dysregulation of SIRT-1 in skeletal muscle from aged mice reduces skeletal muscle performance due to higher PARP-1 activity via GCN5 mediated acetylation. ROS, reactive oxygen species; NAD, nicotinamide adenine dinucleotide; Ac, acetylation; P, protein.

  
  
  

• Research Paper Volume 6, Issue 9 pp 755-769

  Rapamycin treatment of Mandibuloacral Dysplasia cells rescues localization of chromatin-associated proteins and cell cycle dynamics

  Relevance score: 6.7228556

  Vittoria Cenni, Cristina Capanni, Elisabetta Mattioli, Marta Columbaro, Manfred Wehnert, Michela Ortolani, Milena Fini, Giuseppe Novelli, Jessika Bertacchini, Nadir M. Maraldi, Sandra Marmiroli, Maria Rosaria D'Apice, Sabino Prencipe, Stefano Squarzoni, Giovanna Lattanzi

  Keywords: Mandibuloacral Dysplasia (MADA), Prelamin A, SIRT-1, Oct-1, Rapamycin

  Published in Aging on July 19, 2014

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  Lamin A is a key component of the nuclear lamina produced through post-translational processing of its precursor known as prelamin A. LMNA mutations leading to farnesylated prelamin A accumulation are known to cause lipodystrophy, progeroid and developmental diseases, including Mandibuloacral dysplasia, a mild progeroid syndrome with partial lipodystrophy and altered bone turnover. Thus, degradation of prelamin A is expected to improve the disease phenotype. Here, we show different susceptibilities of prelamin A forms to proteolysis and further demonstrate that treatment with rapamycin efficiently and selectively triggers lysosomal degradation of farnesylated prelamin A, the most toxic processing intermediate. Importantly, rapamycin treatment of Mandibuloacral dysplasia cells, which feature very low levels of the NAD-dependent sirtuin SIRT-1 in the nuclear matrix, restores SIRT-1 localization and distribution of chromatin markers, elicits release of the transcription factor Oct-1 and determines shortening of the prolonged S-phase. These findings indicate the drug as a possible treatment for Mandibuloacral dysplasia.

  

  HEK293 cells were transiently transfected with prelamin A constructs yielding wild-type lamin A (LA-WT), non-farnesylatable unprocessable prelamin A (LA-C661M), or farnesylated (partially processed) prelamin A (LA-L647R). (A) Transfected cells were subjected to Chloroquine diphosphate (chloroquine) to block lysosome-mediated degradation. Proteins separated on 8% SDS-PAGE were subjected to Western blot analysis. LA-C661M was accumulated in chloroquine-treated cells, indicating lysosomal degradation of non-farnesylated prelamin A. The presence of LC3-B2 band indicates activation of the lysosomal degradation pathway. (B) Phosphorylation of Serine 404 (P-Ser404) is detected in LA-WT and LA-C661M, while LA-L647R is minimally phosphorylated. LA-S404A is a non-phosphorylatable lamin A mutant and was tested as a negative control for anti-P-Ser404 antibody, LA-S404D is a phosphomimetic mutant for P-Ser404. Densitometric values of P-Ser404 labeled bands normalized to FLAG-labeled bands are reported in the graph as mean values of triplicate experiments +/− Standard Deviation. Asterisks indicate significantly different values relative to LA-WT (p< 0.05) determined by Student's T test. (C) To test rapamycin activity on prelamin A mutants, cells were treated with rapamycin (rapamycin) for the indicated time periods. Farnesylated prelamin A (LA-L647R) was selectively degraded (upper panel). Phosphorylation of Serine 404 (P-Ser404) is shown in the lower panel. (D) The densitometric values of anti-prelamin A labeled bands detected in chloroquine or rapamycin-treated cellular lysates were measured. Data are reported as percentage of the densitometric value of each corresponding untreated sample (designated as 100%). The mean values of triplicate experiments +/− Standard Deviation are reported. Actin has been labeled as a loading control. Prelamin A was detected by anti-prelamin A (Santa Cruz Sc-6214) antibody or by anti- FLAG antibody (Sigma-M2).

  
  
  

  

  (A) Cellular lysates from control, RD or MADA fibroblasts were subjected to Western blot analysis to test prelamin A, lamin A, lamin C and LC3B2 levels under different experimental conditions. Actin was labeled as a loading control. Cultured fibroblasts were left untreated or treated with rapamycin and/or chloroquine. Densitometric analysis of lamin A, lamin C and prelamin A immunoblotted bands is shown (dark red bars highlight untreated samples). Statistically significant differences (p<0.05), measured by the Mann-Whitney test, are indicated by asterisks. Prelamin A was detected by anti-prelamin A antibody (Santa Cruz Sc-6214), lamin A/C by anti-lamin A/C antibody (Santa Cruz Sc-6215). (B) Prelamin A immunofluorescence staining performed using anti-prelamin A 1188-1 antibody, which labels non-farnesylated prelamin A, was detected using a TRITC-conjugated secondary antibody (red). (C) Prelamin A immunofluorescence staining performed using anti-prelamin A 1188-2 antibody, which labels farnesylated prelamin A, was detected using a FITC-conjugated secondary antibody (green). Control, RD or MADA fibroblasts were left untreated (−) or treated with rapamycin (+) for 4 days. Bar, 10 μm. Statistical evaluation of non-farnesylated and farnesylated prelamin A-labeled nuclei is reported in the graphs in (B) and (C), respectively. 200 nuclei per sample were counted. Western blots showing non farnesylated and farnesylated prelamin A on the same treated or untreated cultures are also shown in (B) and (C), respectively. Densitometric values of prelamin A bands in rapamycin-treated samples are reported below each immunoblotted band as percentage of the intensity detected in the corresponding untreated sample. (D) Lamin A/C staining of control, RD and MADA fibroblasts showing misshapen nuclei in laminopathic cells left untreated (untreated) or subjected to 4 day rapamycin treatment (rapamycin). The quantitative analysis is reported in the graph. Statistically significant differences (p<0.05) relative to untreated corresponding samples were calculated using the Mann-Whitney non-parametric test and are indicated by asterisks.

  
  
  

  

  (A) Control, RD and MADA cells left untreated (untreated) or after rapamycin treatment (rapamycin) were stained for LAP2alpha. Levels of LAP2alpha fluorescence intensity and the percentage of nuclei with LAP2alpha mislocalized in nuclear clusters are reported in the upper and lower graph respectively as mean values +/− standard deviation. (B) Control, RD and MADA cells left untreated (untreated) or after rapamycin treatment (rapamycin) were stained for Oct-1. Arrows indicate Oct-1 nuclear foci, arrowheads indicate recovered Oct-1 localization in the nucleoplasm. The pseudo-coloring of Oct-1 pictures obtained by using the Photoshop 7 color mapping function reveals the accumulation of Oct-1 in nuclear foci and at the periphery and recovery by rapamycin treatment in MADA. The intensity of fluorescence is represented on a pseudocolor scale (palette bar). The percentage of nuclei with Oct-1 mislocalized in nuclear clusters is reported in the graph as mean values +/− standard deviation. (C) Proximity Ligation Assay (PLA) between prelamin A and Oct-1. Control and MADA fibroblasts left untreated or treated with rapamycin, were labeled with anti-Oct-1 and anti-prelamin A (SC - 6214) antibodies and probed with Duolink (Sigma) detection reagents according to the manufacturer. Nuclei were counterstained with DAPI. The PLA signals (in red) were counted with the Duolink ImageTool software and the average number of spots in the nucleus per cell (200 cells per sample were counted) is presented in the graph. Statistically significant differences (p<0.05) are indicated by asterisks.

  
  
  

  

  (A) Western blot analysis of SIRT-1 in control and MADA lysates left untreated or after rapamycin treatment. The lysates were obtained in SDS buffer (insoluble) or in RIPA buffer (soluble) to evaluate the amount of SIRT-1 in different nuclear platforms; densitometric values of immunoblotted SIRT-1 bands are shown in the graph as means +/− standard deviation. Statistically significant differences (p<0.05) measured by the Mann-Whitney test are indicated by asterisks. (B) SIRT-1 staining in control or MADA cells left untreated (untreated) or subjected to rapamycin treatment (rapamycin). Non-extracted nuclei (−), nuclei subjected to detergent extraction (NP40) or to DNase treatment and high salt extraction (DNase) are shown. Nuclei were counterstained with DAPI. Scale bar, 10 μm. (C, D) Control, RD and MADA cells left untreated (untreated) or after rapamycin treatment (rapamycin) were stained for trimethyl-H3K9 (tri-H3K9) or acetyl-H3K9. Tri-H3K9 antibody labeling was revealed by FITC-conjugated secondary antibody (green), acetyl-H3K9 antibody labeling was revealed by TRITC-conjugated secondary antibody (red). (E) Tri-H3K9 and acetyl-H3K9 mean fluorescence intensity values measured by the NIS software in 200 nuclei are plotted. Statistically significant differences (p<0.05) are indicated by asterisks. Black asterisks indicate significance versus control cell cultures, red asterisks indicate significance versus the corresponding untreated sample. (F) Western blot analysis of trimethyl-H3K9 (tri-H3K9) and acetyl-H3K9 in control (ctrl) or MADA fibroblasts (MADA) before or after rapamycin treatment. Total H3 is reported as a loading control. The densitometric analysis of immunoblotted tri-H3K9 and acetyl-H3K9 bands is reported in the graph as mean values +/− standard deviation of the mean obtained in three different experiments. Statistically significant differences (p<0.05) measured by the Mann-Whitney test are indicated by asterisks.

  
  
  

  

  (A) Proliferation rate of control or MADA cells left untreated or treated with rapamycin. 1,45 × 10⁵ cells were plated at T0. After the treatment (T4), living cells were counted with trypan blue by a Neubauer camera. (B) Cell cycle analysis of cells left untreated or treated with rapamycin. The acquired FACS data were analyzed by ModFit LT software (Verity Software House, Inc.). Percentage of cells in cell cycle phases (G1, S, G2) is reported in the boxed areas within each panel. Ctrl, control. (C) Relative percentage of cells in each cell cycle phase before and after rapamycin treatment (rapamycin). (D) BrdU was incorporated into living fibroblasts for 4 hours and cells were fixed and subjected to IF staining using anti-BrdU antibody. The percentage of positive nuclei is indicated in the graph as mean value of three different counts obtained in different experiments. Data are means of three different counts +/− standard deviation of the mean. (E) Percentage of PCNA-positive MADA nuclei showing diverse staining patterns (S-phase, G1/G2 pattern). Subconfluent MADA fibroblasts left untreated (untreated) or treated with rapamycin (rapamycin) were immunolabeled for PCNA using anti-PCNA PC10 antibody. The number of nuclei with S-phase or G1/G2 staining pattern or negative for PCNA labeling was determined by counting 200 nuclei per sample.

  
  
  

• News Volume 2, Issue 4 pp 183-184

  Taming the beast within: resveratrol suppresses colitis and prevents colon cancer

  Relevance score: 5.3711004

  Lorne J. Hofseth, Udai P. Singh, Narendra P. Singh, Mitzi Nagarkatti, Prakash S. Nagarkatti

  Keywords: Inflammation, complementary and alternative medicine, SIRT-1, NF-kappaB, inflammatory bowel disease

  Published in Aging on April 30, 2010