Expression levels of miR-34a, miR-181a (C), superoxide dismutase (SOD-2), catalase (CAT), nuclear factor erythroid 2 like 2 (NRF2) (D) and B-cell lymphoma (BCL2) (F) by real-time PCR. gene and protein expression in OA chondrocytes. All the visfatin-caused effects were suppressed by using miR-34a and miR-181a inhibitors. Pre-incubation with BAY-11-7082 counteracted visfatin-induced expression of miRNA, BCL2, SOD-2, CAT and NRF2. Inhibition of miR-34a and miR-181a significantly reduced the activation of p50 NF-B. Visfatin confirms its ability to induce apoptosis and oxidative stress in human OA chondrocytes; these effects appeared mediated by miR-34a and miR-181a via NF-B pathway. We highlight the relevance of visfatin as potential therapeutic target for OA treatment. for 5 min. After centrifugation step, pellets were resuspended in saline solution before analysis at flow cytometry. A density of 1 1 104 cells per assay (a total of 10,000 events) were measured by the instrument and data were analysed with CellQuest software (Version 4.0, Becton Dickinson, San Jose, CA, USA). Results were expressed as median of fluorescence (AU) and indicated as mean of three independent experiments (mean SD). 2.7. Western Blot OA chondrocytes at first passage were seeded in Petri dishes (35 10 mm) at Rabbit Polyclonal to B-RAF a starting density of 1 1 105 cells/chamber in DMEM supplemented with 10% FBS for 24 h. After this period, the medium was removed and the cells were cultured in DMEM with 0.5% FBS for the experiment. After treatment, samples were collected and total cell lysates were obtained with M-PER? Mammalian Protein Extraction Reagent (Thermo-Fisher Scientific, Rockford, IL, Nipradilol USA) containing a protease inhibitor cocktail (Sigma-Aldrich S.r.l., Milan, Italy). Protein concentration was determined by the Bradford method (Bio-Rad Laboratories S.r.l., Milan, Italy). For each experimental condition ten micrograms were loaded into 10% sodium dodecyl sulphate-polyacrylamide electrophoresis gels and separated by molecular size. Proteins were then transferred to a nitrocellulose membrane and, after blocking step, incubated at 4 C overnight with mouse monoclonal anti-BCL2 (sc-509), anti-SOD-2 (sc-130345), anti-CAT (sc-271358) and NRF2 (sc-81342) (Santa Cruz Biotechnology, Italy). Afterwards, the samples were incubated with secondary goat anti-mouse IgG (H+L)-HRP conjugate antibody (1:5000) (Bio-Rad Laboratories S.r.l., Milan, Italy); the reaction was assessed by chemiluminescence (Bio-Rad Laboratories S.r.l., Milan, Italy). The blots were re-probed with HRP-conjugated -actin (Sigma-Aldrich S.r.l., Milan, Italy) used as the loading control. Images of the bands were digitized and the densitometric quantification of the bands was performed by Image-J software (LOCI, University of Wisconsin-Madison, Madison, WI, USA). Results were normalized with the relative loading control. 2.8. Immunofluorescence Analysis Human OA chondrocytes were plated in coverslips in Petri dishes (35 10 mm) at a starting low density Nipradilol of 4 104 cells/chamber, to prevent possible cell overlapping and re-suspended in DMEM supplemented with 10% FBS for 24 h until 80% of confluence. The cells were processed after 24 h of transfection with miR-34a and miR-181a specific inhibitors to evaluate the potential activation of the NF-B pathway. Chondrocytes were washed in PBS and then fixed in 4% paraformaldehyde (ThermoFisher Scientific, Milan, Italy) (pH 7.4) for 15 min at room temperature. Afterwards, the cells were permeabilized with a blocking solution, constituted by PBS, 1% bovine serum albumin (BSA) (SigmaCAldrich, Milan, Italy) and 0.2% Triton X-100 (ThermoFisher Scientific, Milan, Italy), for 20 min at room temperature. Then, chondrocytes were incubated overnight at 4 C with mouse monoclonal anti-p50 subunit primary antibody (sc-8414, Santa Cruz Biotechnology, Italy) (dilution 1:100) dissolved in PBS, 1% BSA and 0.05% Triton X-100. After this period, coverslips were washed three times in PBS and incubated for 1 h with Nipradilol goat anti-mouse IgG-Texas Red conjugated antibody (Southern Biotechnology, Italy) (dilution 1:100) dissolved in PBS, 1% BSA and 0.05% Triton X-100. Finally, after three washes in PBS the coverslips were submitted to nuclear counterstain by 4,6-diamidino-2-phenylindole (DAPI) and then mounted with Vecta shield (Vector Labs). Fluorescence was examined under an Zeiss Axiovert (Zeiss, Germany) light microscope equipped with epifluorescence at 200 and 400 magnification. Immunoreactivity of p50 was semi-quantified as mean densitometric area of p50 signal at nuclear and cytoplasmic level, by AxioVision 4.6 software measure program [17]. At least 100 chondrocytes from each group were analysed. 2.9. Statistical Analysis Three independent experiments were carried out and the results were expressed as the mean SD of triplicate values for each experiment. Data normal distribution was evaluated by ShapiroCWilk, DAgostino and Pearson and KolmogorovCSmirnov tests. Flow cytometry and western blot results were analysed by ANOVA with Bonferroni post-hoc test. Quantitative real-time PCR data were evaluated by one-way ANOVA with a Tukeys post-hoc test using 2?CT values for each sample. All analyses were performed through the SAS System (SAS Institute Inc., Cary, NC, USA) and GraphPad Prism 6.1. A 0.01, Figure 1A) and an increase of superoxide anion production ( 0.01, Figure 1B) in comparison to basal condition. Open in a.