AntimiR-30b Inhibits TNF-α Mediated Apoptosis and Attenuated Cartilage Degradation through Enhancing Autophagy Cell Physiol Biochem 2016;40:883-894 - DOI:10.1159/000453147 Fig. 1. Decreased cell viability and increased apoptosis in response to elevated exposure of ATDC5 cells to TNF-α. ATDC5 cells were plated in 96-well plates at a density of 1×105 cells/well and then cultured in complete medium in the presence or absence of TNF-α (0-20 ng/ml). A. CCK-8 assay to quantify viable cells. The absorbance of the cell culture at OD450 was tested at each time point (6, 12, 24 and 48 h). B. Hoechst staining of cellular DNA and nuclei at 24 h; C. Western blot analysis of the expression of cleaved caspase 3 and cleaved caspase 9 in cells exposed to TNF-α (20 ng/ml) for 6, 12 or 24 h. Control cells cultured in the absence of TNF-α for 24 h were included. GAPDH expression was detected as an endogenous control; D. Quantification of the western blot data by densitometric analysis. All data are presented as the mean ± SD from three independent replicate experiments. *P < 0.05, **P < 0.01 vs. control, untreated cells. © 2016 The Author(s) Published by S. Karger AG, Basel - CC BY-NC-ND 4.0

AntimiR-30b Inhibits TNF-α Mediated Apoptosis and Attenuated Cartilage Degradation through Enhancing Autophagy Cell Physiol Biochem 2016;40:883-894 - DOI:10.1159/000453147 Fig. 2. Changes in autophagy in TNF-α-induced ATDC5 cells. ATDC5 cells were divided into four treatment groups: control (no treatment), 3-MA (treated with 5 mM 3-MA), Rap (treated with 2 µM Rap), TNF-α (treated with 20 ng/mL TNF-α). A. ATDC5 cells were transfected with GFP-LC3B-expressing plasmid and treated as indicated. Autophagosome formation was indicated by GFP-labelled puncta; the quantitative results are shown. B. Autophagy was assessed by western blot analysis of the autophagy-related proteins BECN1, ATG5, LC3B-II and p62; densitometric analysis of the protein bands is shown. The data are presented as the mean ± SD from three independent replicate experiments. *P < 0.05, **P < 0.01 vs. control cells. © 2016 The Author(s) Published by S. Karger AG, Basel - CC BY-NC-ND 4.0

AntimiR-30b Inhibits TNF-α Mediated Apoptosis and Attenuated Cartilage Degradation through Enhancing Autophagy Cell Physiol Biochem 2016;40:883-894 - DOI:10.1159/000453147 Fig. 3. MiR-30b directly targets BECN1 and ATG5. A. Sequence of the putative miR-30b binding site within the 3ʹ-UTR of BECN1 and ATG5 mRNA. B. A luciferase reporter assay was performed by cotransfecting cells with wild-type BECN1 or ATG5 3ʹ-UTR (BECN1-WT or ATG5-WT) reporter plasmids or mutated BECN1 or ATG5 3ʹ-UTR (BECN1-Mut or ATG5-Mut) reporter plasmids, and miR-30b or nonspecific control miRNA (NC). Cells transfected with only BECN1 or ATG5 or BECN1-Mut or ATG5-Mut were used as controls. Fluorescence intensity was measured. C. RT-PCR analysis of the relative mRNA expression levels of BECN1 or ATG5 in cells transfected with miR-NC, miR-30b, antimiR-NC or antimiR-30b. D. Western blot analysis of the protein expression levels of BECN1 or ATG5 in cells transfected with miR-NC, miR-30b, antimiR-NC or antimiR-30b. **P < 0.01 vs. control cells. © 2016 The Author(s) Published by S. Karger AG, Basel - CC BY-NC-ND 4.0

AntimiR-30b Inhibits TNF-α Mediated Apoptosis and Attenuated Cartilage Degradation through Enhancing Autophagy Cell Physiol Biochem 2016;40:883-894 - DOI:10.1159/000453147 Fig. 4. MiR-30b regulates autophagy and the expression of autophagy genes in ATDC5 cells. ATDC5 cells (treated with 20 ng/mL TNF-α) were divided into seven groups: control, 3-MA (treated with 5 mM 3-MA), Rap (treated with 2 µM Rap), miR-NC (co-transfected with miR-NC), miR-30b (co-transfected with miR-30b), antimiR-NC (co-transfected with antimiR-NC), and antimiR-30b (co-transfected with antimiR-30b). A. ATDC5 cells were transfected with GFP-LC3B-expressing plasmid and treated as indicated. Autophagosome formation was indicated by GFP-labelled puncta; the quantitative results are shown. B. Autophagy was assessed by western blot analysis of LC3-II and P62; densitometric analysis of the protein bands is shown. The data are presented as the mean ± SD from three independent replicate experiments. **P < 0.01 vs. control. © 2016 The Author(s) Published by S. Karger AG, Basel - CC BY-NC-ND 4.0

AntimiR-30b Inhibits TNF-α Mediated Apoptosis and Attenuated Cartilage Degradation through Enhancing Autophagy Cell Physiol Biochem 2016;40:883-894 - DOI:10.1159/000453147 Fig. 5. Anti-miR-30b repressed TNF-α-induced apoptosis by enhancing autophagy. A. ATDC5 cells, treated with 20 ng/mL TNF-α, were divided into six groups: 3-MA (treated with 5 mM 3-MA), Rap (treated with 2 µM Rap), miR-NC (transfected with miR-NC), miR-30b (transfected with miR-30b), antimiR-NC (transfected with antimiR-NC) and antimiR-30b (transfected with antimiR-30b). Untreated cells and cells treated with 20 ng/mL TNF-α only were included as controls. A CCK8 assay was performed and the apoptotic index was determined for each treatment group. **P < 0.01 vs. control (20 ng/mL TNF-α); ##P < 0.01 vs. control (no treatment). B. ATDC5 cells treated with 20 ng/mL TNF-α and transfected with miR-NC, miR-30b, antimiR-NC or antimiR-30b, were subjected to western blot analysis to analyse the expression of cleaved caspase 3 and 9. NAPDH expression was also analysed as an endogenous control. C. Quantitative analysis of the western blot data showing the relative protein expression levels. The data are presented as the mean ± SD from three independent replicate experiments. **P < 0.01 vs. control. © 2016 The Author(s) Published by S. Karger AG, Basel - CC BY-NC-ND 4.0

AntimiR-30b Inhibits TNF-α Mediated Apoptosis and Attenuated Cartilage Degradation through Enhancing Autophagy Cell Physiol Biochem 2016;40:883-894 - DOI:10.1159/000453147 Fig. 6. AntimiR-30b effected TNF-α-induced ECM degradation by enhancing autophagy. A, ATDC5 cells were divided into two groups: control (no treatment) and 20 ng/ml TNF-α. B. ATDC5 cells (treated with 20 ng/mL TNF-α) were divided into five groups: control (TNF-α only), miR-NC (transfected with miR-NC), miR-30b (transfected with miR-30b), antimiR-NC (transfected with anti-miR-NC), and anti-miR-30b (transfected with antimiR-30b). *P < 0.05 vs. control cells C. ATDC5 cells (treated with 20 ng/mL TNF-α) were divided into four groups: antimiR-NC (treated or untreated with 5 mM 3-MA), anti-miR-30b (treated or untreated with 5 mM 3-MA). Forty-eight hours after treatment, the cells were subjected real-time RT-PCR to determine the mRNA levels of ACAN, Col2A, sox9 and MMP13 in these cells. D. ATDC5 cells (treated with 20 ng/mL TNF-α) were divided into two groups: antimiR-NC or anti-miR-30b. Forty-eight hours after treatment, the cells were subjected real-time RT-PCR to determine the mRNA levels of MMP2, ADAMTS-1, ADAMTS-4, TIMP-1 and TIMP-3 in these cells. Data are presented as the mean ± SEM. *P < 0.05 vs. anti-miR-NC cells, #P < 0.05 vs. anti-miR-30b. © 2016 The Author(s) Published by S. Karger AG, Basel - CC BY-NC-ND 4.0