Hydroxytyrosol modulates the levels of microRNA-9 and its target sirtuin-1 thereby counteracting oxidative stress-induced chondrocyte death  S. D'Adamo, S. Cetrullo, S. Guidotti, R.M. Borzì, F. Flamigni  Osteoarthritis and Cartilage  Volume 25, Issue 4, Pages 600-610 (April 2017) DOI: 10.1016/j.joca.2016.11.014 Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

Fig. 1 Opposite variations of SIRT-1 and miR-9 levels in response to H2O2 and HT treatments in human primary and C-28/I2 chondrocytes. Cell cultures were pre-incubated in the absence or in the presence of 100 μM HT for 30 min before addition of 100 μM H2O2. After 24 h of incubation cells were harvested for SIRT1 detection by western blotting. Representative images and relative quantification for SIRT1/β-actin ratio are shown (A). After 4 h of incubation samples were collected for miR-9 quantification by qPCR analysis (B). Site of matching between SIRT1 3′UTR and miR-9 sequences (provided by TargetScan database) is shown (C). Values are expressed as 95% confidence intervals, and P values of the differences are shown above comparisons between groups. Osteoarthritis and Cartilage 2017 25, 600-610DOI: (10.1016/j.joca.2016.11.014) Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

Fig. 2 Impact of the miR-9 silencing on the toxicity of H2O2 in human primary chondrocytes. Primary cells were transfected for 24 h with antimiR-9 or antimiR-NC (50 nM). After 4 h incubation cells were harvested and miR-9 levels were determined by qPCR (A). After 24 h incubation with or without H2O2, cells were collected for SIRT1 detection by western blotting. Representative images and relative quantification of SIRT1/β-actin ratio are shown (B). Alternatively, cells were counted to assess cell viability by trypan blue exclusion test or harvested and analyzed for caspase activity (C). In addition, cells were transfected for 24 h with antimiR-9 or antimiR-NC (50 nM) and siCTRL or siSIRT-1 (25 nM). After 24 h incubation with or without 100 μM H2O2, cells were counted to assess cell viability by trypan blue exclusion test or harvested and analyzed for caspase activity (D). Values are expressed as 95% confidence intervals, and P values of the differences are shown above comparisons between groups. Osteoarthritis and Cartilage 2017 25, 600-610DOI: (10.1016/j.joca.2016.11.014) Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

Fig. 3 Impact of premiR-9 transfection on the HT-mediated protection in human primary chondrocytes. Cells were transfected for 24 h with premiR-9 or premiR-NC (50 nM). After 4 h incubation with 100 μM H2O2 or 100 μM HT cells were harvested and miR-9 levels were determined by qPCR (A). After 24 h incubation with or without HT, cells were collected for SIRT-1 detection by western blotting. Representative images and relative quantification for SIRT-1/β-actin ratio are shown (B). Alternatively after 24 h incubation with 100 μM H2O2 or 100 μM HT, cells were counted to assess cell viability by trypan blue exclusion test or harvested and analyzed for caspase activity (C). Values are expressed as 95% confidence intervals, and P values of the differences are shown above comparisons between groups. Osteoarthritis and Cartilage 2017 25, 600-610DOI: (10.1016/j.joca.2016.11.014) Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

Fig. 4 Impact of antimiR-9 and premiR-9 transfection on H2O2-induced cell death and HT-mediated protection in C-28/I2 cells. Cells were transfected for 24 h with antimiR-9 or antimiR-NC (50 nM) and premiR-9 or premiR-NC (50 nM). After 4 h incubation with 100 μM H2O2 or 100 μM HT cells were harvested and miR-9 levels were determined by qPCR (A). After 24 h incubation with or without HT, cells were collected for SIRT1 detection by western blotting. Representative images and relative quantification for SIRT1/β-actin ratio are shown (B). Alternatively after 24 h incubation with 100 μM H2O2 or 100 μM HT, cells were counted to assess cell viability by trypan blue exclusion test (C). In addition, cells were transfected for 24 h with antimiR-9 or antimiR-NC (50 nM) and siCTRL or siSIRT1 (25 nM). After 24 h incubation with or without 100 μM H2O2, cells were counted to assess cell viability by trypan blue exclusion test (D). Values are expressed as 95% confidence intervals, and P values of the differences are shown above comparisons between groups. Osteoarthritis and Cartilage 2017 25, 600-610DOI: (10.1016/j.joca.2016.11.014) Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

Fig. 5 SIRT1 is a direct target of miR-9 in C-28/I2 cells. Three different sequences were designed and provided within pEZX-MT06 reporter vector (Genecopoeia); a first sequence deleted of seed sequences (3′UTR mut1); a second one deleted of miR-9 3′ pairing sequence as well as of seed sequence (3′UTR mut2); a last one with the full 3′UTR sequence (3′UTR wt) (A). Cells were co-transfected with either plasmid carrying 3′UTR mut1, 3′UTR mut2 or 3′UTR wt, with premiR-9 or premiR-NC. The dual luciferase activity of the transfected cells was detected and the luciferase activity was normalized on renilla activity (B). Values are expressed as 95% confidence intervals, and P values of the differences are shown above comparisons between groups. Osteoarthritis and Cartilage 2017 25, 600-610DOI: (10.1016/j.joca.2016.11.014) Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

Fig. 6 MiR-9 levels influence the expression of OA-related genes in primary OA chondrocytes. Cells were transfected for 24 h with antimiR-9 or antimiR-NC (50 nM) and premiR-9 or premiR-NC (50 nM). After 4 h incubation with 100 μM H2O2 or 100 μM HT cells were harvested and analyzed by qRT-PCR for the amount of MMP-13 mRNA, VEGF mRNA and RUNX-2 mRNA. Values are expressed as 95% confidence intervals, and P values of the differences are shown above comparisons between groups. Osteoarthritis and Cartilage 2017 25, 600-610DOI: (10.1016/j.joca.2016.11.014) Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

Supplementary Fig. 1 SIRT-1 knockdown in human primary chondrocytes. Cells were transfected for 24 h with siCTRL or siSIRT1 (25 nM) and then harvested for SIRT-1 detection by western blotting. Representative image and relative quantification of SIRT-1/β-actin ratio are shown. Values are expressed as 95% confidence intervals, and P value of the difference is shown above comparison between groups Osteoarthritis and Cartilage 2017 25, 600-610DOI: (10.1016/j.joca.2016.11.014) Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions