Additional file 2: of Lymphocyte opioid receptors as innovative biomarkers of osteoarthritic pain, for the assessment and risk management of opioid tailored therapy, before hip surgery, to prevent chronic pain and opioid tolerance/addiction development: OpMarkArt (Opioids-Markers-Arthroprosthesis) study protocol for a randomized controlled trial

Numeric pain rating scale. (PDF 180 kb

sj-docx-1-tab-10.1177_1759720X231214903 – Supplemental material for Effects of adipose-derived mesenchymal stem cell conditioned medium on human tenocytes exposed to high glucose

Supplemental material, sj-docx-1-tab-10.1177_1759720X231214903 for Effects of adipose-derived mesenchymal stem cell conditioned medium on human tenocytes exposed to high glucose by Maria Consiglia Trotta, Annalisa Itro, Caterina Claudia Lepre, Marina Russo, Francesca Guida, Antimo Moretti, Adriano Braile, Umberto Tarantino, Michele D’Amico and Giuseppe Toro in Therapeutic Advances in Musculoskeletal Disease</p

Time-course of 26S proteasome activity over the HSR and Western blotting analyses.

<p>(A) The chymotryptic-like activity of 26S proteasome particles partially purified from <i>wild-type</i> (black square), treated (red circle) and in presence of 10–40 nanomol/L exogenous IDE added to extracts from treated cells (green triangle), was assayed through the proteasome assay: the relative activity represents the ratio between the velocity of degradation of 200 micromol/L fluorogenic substrate at time 0 (resting conditions) <i>vs</i> the indicated time of recovery. The reaction was followed over 3 h of incubation at 37°C. Results presented are the means +/- S.E. of five independent experiments* (<i>p</i><0.05, one way ANOVA, followed by Tukey’s test, <i>n</i> = 15). Western blotting analysis of crude cell extracts: (B) Filters were probed with antibodies specific for: IDE (110 kDa), 26S proteasome (22 kDa), HSP70 (70 kDa), GAPDH (37 kDa). (C) Filters were probed with a polyclonal antibody specific for ubiquitin. A representative immunoblot of five independent experiments is shown.</p

26S proteasome activity in crude cell extracts from treated and <i>wild-type</i> SHSY5Y.

<p>The proteasome assay on crude cell extracts from either treated or <i>wild-type</i> cells was performed 48 h and 72 h after the anti-sense oligonucleotide delivery. The chymotryptic-like (A), the tryptic-like (B) and the caspase-like (C) activities of the 26S proteasome particles were assayed on specific fluorogenic substrates. Values reported are the means +/- S.E. of five independent experiments. *, significantly different from control (<i>p</i><0.05, one way ANOVA, followed by Tukey’s test, <i>n</i> = 15).</p

Confocal images of control and stressed fixed SHSY5Y cells 24 h after heat administration.

<p>Cell localization of IDE was determined using anti-IDE mouse monoclonal (A and B) or anti-IDE rabbit polyclonal (C) antibodies. The Endoplasmic Reticulum was stained with anti-calnexin (A), while Golgi was labelled with anti-giantin rabbit polyclonal antibody (B). Plasma membrane was stained with anti-Na⁺/ K⁺-ATPase mouse monoclonal antibody (C) Nuclei were counterstained with DAPI (blue)(A,B). Bars correspond to 20 micrometers.</p

Biochemical characterization of IDE-26S proteasome interaction.

<p>The chymotriptic-like (A), tryptic-like (B) and caspase-like (C) activities of a highly purified 26S proteasome were assayed on specific fluorogenic substrates in presence of highly purified eukaryotic IDE. The reported values represent the ratio between the velocity of degradation of 50 micromol/L fluorogenic substrate by 1 nM 26S in the absence and the presence of different IDE concentrations, ranging from 10 nanomol/L to 50 nanomol/L. Results presented are the means +/- S.E. of five independent experiments.</p

LC-MS/MS analysis of the (613–651) peptide incubated with MMP-2.

<p>Chromatographic profiles of the (613–651) peptide incubated for 5 hours without (<b>A</b>) or with (<b>B</b>) MMP-2. MS/MS spectra of the (636–651) peptide eluting at RT 18.9 min (<b>C</b>) and (619–629) peptide eluting at RT 23.4 min (<b>D</b>). Matched b and y ions are colored in red and blue, respectively, while precursor ions loosing H₂O or NH₃ are indicated in green.</p

The enzymatic processing of α-dystroglycan by MMP-2 is controlled by two anchoring sites distinct from the active site - Fig 2

<p><b>The αDG (613–651) peptide affects the degradation of αDG (483–628) peptide</b> (<u><i>Panel A</i></u>) Aminoacidic sequences associated with each subunits of murine DG and its schematic representation. Enzymatic proteolysis profiles of the purified α-DG C-terminal domain (483–628) construct by MMP-2 (<u><i>Panel B</i></u>), or by cdMMP-2 (<u><i>Panel C</i></u>) are represented by the sodium dodecyl sulfate polyacrylamide gels stained with Coomassie blue. Four-point time course was represented: time 0, time 30 s, time 1 hour, time 5 hours in the absence (upper panel) and in the presence (lower panel) of 4 μM of the α-DG (613–651) peptide. Equal aliquots from the incubation mixtures were collected at increasing times, the reaction was stopped and samples were separated by TRIS glycine 10–20% precast gel.</p

The αDG (613–651) peptide modulation of MMP-2 catalysis.

<p>Dependence on the α-DG(613–651) peptide concentration of <i>k</i>_<i>cat</i> (<u><i>panel A</i></u>), <i>K</i>_<i>m</i> (<u><i>panel B</i></u>) and <i>k</i>_<i>cat</i><i>/K</i>_<i>m</i> (<u><i>panel C</i></u>) for the enzymatic processing of the fluorogenic peptide by whole MMP-2 (o) and by cdMMP-2 (x) at pH 7.3 and 37°C, as obtained by the analysis of data reported in Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0192651#pone.0192651.g004" target="_blank">4</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0192651#pone.0192651.g005" target="_blank">5</a>. Continuous lines have been obtained by applying Eqs (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0192651#pone.0192651.e002" target="_blank">2</a>) and (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0192651#pone.0192651.e003" target="_blank">3</a>), employing parameters reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0192651#pone.0192651.t001" target="_blank">Table 1</a>. Arrows indicate the value of the parameter in the absence of the α-DG(613–651) peptide, which is represented as “o” for the intact MMP-2 and as “x” for cdMMP-2.</p

The effect of αDG (613–651) peptide on the catalytic parameters for the MMP-2 proteolysis.

<p>Lineweaver-Burk plot of the enzymatic activity of 60 nM MMP-2 at 37°C at pH 7.3 as a function of the fluorogenic substrate concentration at different concentrations of α-DG(613–651) peptide, namely (<u><i>panel A</i></u>) 0 (o), 23 nM (x), 125 nM (*), 200 nM (Δ) and 0.4,μM (25CA), and (<u><i>panel B</i></u>) 0.4 μM (o), 1 μM (x), 2 μM (*) and 4 μM (◊). Continuous lines have been obtained by applying Eqs. (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0192651#pone.0192651.e001" target="_blank">1</a>)–(<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0192651#pone.0192651.e003" target="_blank">3</a>), employing parameters reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0192651#pone.0192651.t002" target="_blank">Table 2</a>. (<u><i>Panel C</i></u>): namely 0 (o), 23 nM (x), 60 nM (*) and 0.2 μM (◊), and, (<u><i>panel D</i></u>) 0.2 μM (o), 0.6 μM (x) and 2 μM (*) and, (<u><i>panel D</i></u>) 0.2 μM (o), 0.6 μM (x) and 2 μM (*). Continuous lines have been obtained by applying Eqs. (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0192651#pone.0192651.e001" target="_blank">1</a>)–(<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0192651#pone.0192651.e003" target="_blank">3</a>), employing parameters reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0192651#pone.0192651.t002" target="_blank">Table 2</a>. Dashed line corresponds to the Lineweaver-Burk plot of the enzymatic activity of whole MMP-2 in the absence. Dashed-dotted lines corresponds to the Lineweaver-Burk plot in the presence of the α-DG(613–651) peptide 0.2 μM and 2 μM α-DG(613–651) peptide in <i>panel C</i> and <i>panel D</i>, respectively. Where not shown, standard deviation is smaller than symbol.</p