PMC

(A) Relative levels of branched chain amino acids in young (MY) and senescent (MS) myoblasts. (B) Carnitine conjugates of BCAA-derived biochemicals. For details of box plots see Figure legend 4.

(A) Immunocytochemistry against desmin evidences morphological changes in senescent human myoblasts (SEN) when compared to young cells (CPD 30). Note the increased diameter and irregular shapes of the formers, as previously described []. (B) p16 protein levels during replicative senescence in human myoblasts. (C) Densitometric analysis of the p16/emerin ratio showed a significant increase (n=3; *P<0.001) in p16 levels during replicative senescence. P16 protein levels are expressed as relative values and shown as mean ± S.D.

(A) Free fatty acids profiling in young (MY) and senescent myoblasts (MS). (B) Increased glycerolipids turnover in senescent cells. PL: phospholipids; TAG: triacylglycerols; DAG: diacylglycerols; MAG: monoacylglycerols; FA: fatty acids; GP: glycerol phosphate. C: choline; E: ethanolamine. (C) Free carnitine and acylcarnitine profiling. The acyl chain length (c) is denoted by the corresponding metabolite number (e.g., C0 = free carnitine, C2 = acetylcarnitine; C3 =proprionylcarnitine). (D) Sphingolipids metabolism in young and senescent myoblasts. Data are expressed as mean ± S.E.M of six independent experiments. * p <0.05. For details of box plots see Figure legend 4.

Chymotrypsin like (A), trypsin-like (B), and caspase-like (C) peptidase activities of the proteasome were measured during replicative senescence. Protein levels of proteasome catalytic subunits (β1, β2, and β5) were assessed by western blot (D) and catalytic subunits protein levels were quantified by densitometric analysis (E). Quantification of carbonylated proteins (F), proteins modified by different glycated end products (G), or modified by the lipid peroxidation product 4-hydroxynonenal (H) during replicative senescence of human myoblasts. Protein modifications are expressed as relative values and shown as mean±S.D. (n=3). Data were analyzed by two-way ANOVA followed by Bonferroni's post hoc test. * P<0.05.

Cellular protein extracts from young (30 CPD) and senescent human myoblasts were separated by two-dimensional gel electrophoresis. After the second dimension, gels were either stained with colloidal Coomassie Brilliant Blue G (bottom panels) or electrotransferred onto nitrocellulose membranes for subsequently detection of: carbonylated proteins using the OxyBlot^TM kit (A); glycoxidation protein adducts (B) and HNE-modified proteins (C). Presented results are from one representative experiment of three independent experiments using three different batches of cells. Numbers refer to the spots evidenced as consistently increased in senescent cells identified by MS/MS. (D) Venn diagram depicting the distribution of proteins in relation with the modifications studied (see also , and ). (E) Modified proteins were grouped into functional categories through the use of Ingenuity Pathways Analysis. The bars represent the biological functions identified, named in the x-axis. The dotted line represents the threshold above which there are statistically significantly more proteins in a biological function than expected by chance. The identified proteins associated with each pathway are indicated.

(A) Modified enzymes identified in senescent cells and related to the central metabolism are represented in boxes. (B) Altered metabolites of central metabolism profiling in young (30 CPD) (MY) and senescent myoblast (MS). For the box plots, the top and bottom of the boxes represent the 75^th and 25^th percentile, respectively. The solid bar across the box represents the median value, while the + is the mean. Any statistical outliers are represented by a circle. The Y axis is the median scaled value (relative level). The fold change and the corresponding p value in senescent cells relative to their young counterpart is indicated in each plot (see also ). (C) Glucose flux in young and senescent myoblasts measured by [U-¹⁴C] glucose oxidation into ¹⁴CO₂. (D) Oxygen consumption rates (OCR) of young and senescent myoblasts were monitored using the Seahorse Bioscience Extra Cellular Flux Analyzer. Mitochondrial respiration was determined in basal conditions (growth media), in the presence of oligomycin (leak), and finally in the presence of increasing amounts of carbonyl cyanide m-chlorophenylhydrazone (CCCP; 1-30 μM) to determine the maximal respiration rate. The respiration reserve capacity (spare) was calculated by subtracting the basal to the maximal respiration. The OCR values were normalized to cellular size.