Fig. 4.

Kindlin-2 deletion promotes Nlrp3 inflammasome activation, ECM catabolism and cell apoptosis in NP cells in vitro. a Immunohistochemical (IHC) staining of Nlrp3, Caspase-1(Casp1) and IL-1β in human NP samples. Scale bar, 50 μm. b, c IF staining of Nlrp3, Casp1 and IL-1β in NP tissues in lumbar IVDs of control and cKO mice at 8 months of age. Scale bar, 50 μm. d–f Western blotting analyses of K2, Col2a1, Mmp13, Nlrp3, Casp1, and IL-1β in NP cells, which were transfected with negative control siRNA (si-NC) or K2 siRNA (si-K2), negative control plasmid (NC-PL) or K2 plasmid (K2-PL), and then treated with or without compression loading (CL) treatment. n = 3. g The levels of IL-1β in the conditioned media of cultured NP cells treated as in (d), as detected by ELISA. n = 3. h, i TUNEL staining of NP cells, which were treated as in (d). Scale bar, 50 μm. n = 4. j–m Western blotting analyses of K2, Nlrp3, and Casp1 in NP cells, which were treated with different doses (0, 25, 50, 100 ng·mL⁻¹) of IL-1β for 24 h or 50 ng·mL⁻¹ IL-1β for different time (0, 12, 24, 48 h) with or without CL treatment. n = 3. n, o Western blotting analyses of Casp1, IL-1β, Col2a1, and Mmp13 in NP cells, which were transfected with si-NC or si-K2, pretreated with or without MCC950, and then treated with or without CL treatment. n = 3. p The levels of IL-1β in the conditioned media of cultured NP cells treated as in (n), as detected by ELISA. n = 3. q, r Apoptosis rate of NP cells, which were treated as in (n), was measured by flow cytometry analyses. n = 4. NS no statistical significance, *P < 0.05, **P < 0.01, ***P < 0.001