Journal: International Journal of Molecular Sciences

Article Title: Sortilin Is Upregulated in Osteoarthritis-Dependent Cartilage Calcification and Associated with Cellular Senescence

doi: 10.3390/ijms241512343

Figure Lengend Snippet: Sortilin and ALP are upregulated in osteogenically differentiated human chondrocytes. Isolated human chondrocytes were treated with osteogenic medium (OM) for up to 21 days to induce calcification. Chondrocyte medium (CM) was used as a control. ( A ) Quantification of eluted Alizarin Red S bound to chondrocytes after treatment with CM and OM to measure calcification. Treatment with OM leads to significantly increased calcification at days 7 and 21, compared with control (CM) (two-way ANOVA F (1, 20) = 29.25; p < 0.0001). Furthermore, there is a significant increase in calcification over time (two-way ANOVA F (2, 40) = 22.78; p < 0.0001). ( B ) Western blot of sortilin and ALP after osteogenic differentiation. ( C ) Sortilin is significantly increased at day 7 (OM) compared to control (CM). Over time, there continues to be a significant increase in sortilin (two-way ANOVA: F (2, 32) = 13.50; p ≤ 0.0001). ( D ) ALP shows a significant increase in protein levels at day 21 in the OM group compared to the control. (two-way ANOVA F (2, 28) = 4.206; p = 0.0253). ( E ) ALP activity assay of CM and OM chondrocytes. At day 21, the ALP activity of OM chondrocytes is significantly increased compared to CM chondrocytes (two-way ANOVA: F (2, 38) = 10.97; p = 0.0002). For statistical evaluation, a two-way ANOVA with Sidak’s post-hoc test was performed. ns: not significant; * p < 0.05, ** p < 0.01, **** p < 0.0001.

Article Snippet: The membrane was then incubated with anti-sortilin/NT3 polyclonal rabbit antibody (1:1000; Abcam, Cambridge, MA) or anti-ALP (Tissue Non-Specific) polyclonal rabbit antibody (1:1000; GeneTex Inc., Irvine, CA, USA) or anti-GAPDH monoclonal mouse antibody (1:1000; Cell Signailing Technology, Danvers, MA, USA) at 4 °C overnight.

Techniques: Isolation, Western Blot, ALP Activity Assay, Activity Assay

Journal: International Journal of Molecular Sciences

Article Title: Sortilin Is Upregulated in Osteoarthritis-Dependent Cartilage Calcification and Associated with Cellular Senescence

doi: 10.3390/ijms241512343

Figure Lengend Snippet: Sortilin is upregulated by senescence. Human chondrocytes were stimulated with mitomycin C for up to 10 days to induce cellular senescence. ( A ) Gene expression of sortilin after 5 and 10 days of treatment with mitomycin C (RM one-way ANOVA F (1.379, 9.656) = 16.88; p = 0.0013; N = 8). ( B ) Gene expression of p16 after 5 and 10 days of treatment with mitomycin C (RM one-way ANOVA: F (2, 14) = 5.080; p = 0.0219; N = 8). ( C ) Gene expression of p21 after 5 and 10 days of stimulation with mitomycin C (RM one-way ANOVA: F (1.367, 9.571) = 49.96; p < 0.0001). ( D ) Gene expression of ALP after 5 and 10 days of stimulation with mitomycin C (RM one-way ANOVA: F (1.083, 4.331) = 25.42, p = 0.0056). ( E ) Sortilin immunofluorescence showing percent sortilin positive cells as a function of duration of stimulation and representative images of sortilin immunostaining at days 1, 5, and 10 (RM one-way ANOVA: F (1.028, 2.057) = 1.077; p = 0.4092; N = 3). ( F ) p16 immunofluorescence with the percentage of p16 pos. cells depending on the duration of stimulation and representative images of p16 staining at days 1, 5, and 10 (RM one-way ANOVA: F (1.263, 2.527) = 1.153; p = 0.399; N = 3). All pictures are taken at 400× magnification (scale bar 50 µm). * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The membrane was then incubated with anti-sortilin/NT3 polyclonal rabbit antibody (1:1000; Abcam, Cambridge, MA) or anti-ALP (Tissue Non-Specific) polyclonal rabbit antibody (1:1000; GeneTex Inc., Irvine, CA, USA) or anti-GAPDH monoclonal mouse antibody (1:1000; Cell Signailing Technology, Danvers, MA, USA) at 4 °C overnight.

Techniques: Expressing, Immunofluorescence, Immunostaining, Staining