Fig. 1
Cell activation with a TLR-2/6 agonist, caused an increase in the number of senescent cells and SASP factor release in cells from non-degenerate human IVDs. A p16ink4a immunostaining images of untreated cells (a–d) from healthy donor IVDs or treated with either TLR-2/6 agonist (e–h), TLR-1/2 agonist (i–l), or TLR-4 agonist (m–p) cultured for 6, 12, 24, or 48 h. Scale bars = 25 μm. B Quantification of the percentage of p16ink4a positive cells in the control and treated cells for the four time points. Examples of positive cell are indicated by the red arrow and for negative cells by the black arrow. C–E qPCR was performed using cells from healthy donor IVDs cultured for 48 h with TLR-2/6 agonist, TLR-1/2 agonist, or TLR-4 agonist. Gene expression for C TLR-1, -2, -4, and -6; D senescence markers p16ink4a and p21; and E SASP factors (CCL2, CCL5, CCL7, CCL8, IL-6, IL-8, TNF-α, NGF. and BDNF). Fold changes were normalized relative to the non-treated control (CTRL). B–E Two-way ANOVA with Tukey’s multiple comparisons test, n = 5; significance was evaluated between treated groups compared to control group. Data shown as mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001