Il 6 gtx110 527

NP cells were divided into four groups, cultured in TASCL microplates, and treated with and without AdipoRon and/or IL-1β (control, group C; 2 μM AdipoRon, group A; 10 ng/mL IL-1β, group I; 2 μM AdipoRon + 10 ng/mL IL-1β, group A + I). Cell clusters, formed on TASCL 48 h after treatment, were incubated with primary antibodies (1:200) against IVD NP-associated CD24 (sc-11,406; Santa Cruz Biotechnology, Santa Cruz, CA, USA), TNF-α (GTX110,520; GeneTex, San Antonio, TX, USA), IL-6 (GTX110,527; GeneTex), ADAMTS-4 (sc-16,533; Santa Cruz Biotechnology), and MMP-13 (LS-B3168; LifeSpan BioSciences, Seattle, WA, USA) overnight at 4 °C. Cell clusters were further treated with secondary antibodies conjugated with Alexa Fluor 488 (A21200; Thermo Fisher Scientific), Alexa Fluor 568 (A21468; Thermo Fisher Scientific) or Alexa Fluor 647 (A31573; Thermo Fisher Scientific) for 1 h at 25 °C. Finally, cells were counterstained with DAPI (D1306; Thermo Fisher Scientific). Immunofluorescent images of cell clusters on TASCL were obtained using a microscope (BZ-X700; Keyence, Osaka, Japan). Relative fluorescence intensities of 10 randomly selected cell clusters per image were measured using a microscope and BZ-X700 software as previously described [52 (link)] (Figure 9a).

Antibody against p-Stat-3(pS727)(sc-8001) was purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Antibodies against p-Stat-3(pY705)(#9145), p-NFκB-p65(pS536)(#8242), and NFκB -p65(#3033) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Antibodies against Stat-3(GTX15523), IL1-β(GTX22105), TNF-α(GTX110520), and IL-6 (GTX110527) were purchased from GeneTex, Inc. (Irvine, CA, USA). Antibody against visfatin(ab58640) was purchased from Abcam plc. (Cambridge, UK). Antibody against β-actin(A5411) was purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). LHC-9 medium was purchased from Invitrogen (Carlsbad, CA, USA). FK866, lipopolysaccharide, hematoxylin, and eosin were purchased from Sigma-Aldrich (St Louis, MO, USA).