Fig 2: Diseased tendon tissues express markers of stromal fibroblast activation. a mRNA expression of stromal fibroblast activation markers podoplanin (PDPN), CD248 and CD106 in healthy subscapularis tendons and diseased and post-treatment supraspinatus tendons. Bar shows median values. Statistically significant differences were calculated using the Kruskal-Wallis test with the pairwise post hoc Mann-Whitney U test; **p < 0.01, ***p < 0.001. b Representative images of 3,3’-diaminobenzidine immunostaining (brown) for PDPN, CD248 and CD106 in healthy, diseased and post-treatment supraspinatus tendons. Nuclear counterstain is haematoxylin. Scale bar 50 μm. c and d Representative immunofluorescence images of sections of diseased supraspinatus tendons stained for markers of stromal activation (PDPN, green; CD248 and CD106, purple) and toll-like receptor 4 (TLR4) (red). Cyan represents POPO-1 nuclear counterstain. Scale bar 20 μm

Fig 3: Analysis of IL-1β and TNFα regulation of key adhesion molecules involved in leukocyte adhesion. a The flow plots show the cell-surface expression levels of adhesion molecules 24 h after cytokine treatment (5 ng/mL). Of this panel, IL-1β and TNFα treatments appear to substantially increase CD54 (ICAM-1) and CD106 (VCAM-1) expression only. Note also the bimodal distribution for CD106 following IL-1β activation. The black line denotes the autofluorescence of the cells. The blue curve represents the basal level of expression, whereas the red curve is the expression level following cytokine treatment. b Time course of selectin (CD62E, P and L) expression following cytokine activation of the hCMVECs. These data are from a single experiment, which is representative of at least three independent experiments

Fig 4: Expression of CD248 mRNA and protein in healthy and diseased tendon cells after IL-1β treatment. Tendon cells were derived from healthy hamstring (n = 5 donors) and diseased supraspinatus tendons (n = 5 donors). Tendon cells were treated with medium containing vehicle only or IL-1β (10 ng/mL-1) for 24 hours. CD248 mRNA (a) and CD248 protein (b) are shown in healthy and diseased tendon cells after vehicle and IL-1β treatments. c Representative fluorescence-activated cell sorting (FACS) contour plots for CD106 from healthy and diseased tendon cells after vehicle and IL-1β treatments gated on CD45-CD34- cells. Bar shows median values. Statistically significant differences were calculated using the Kruskal-Wallis test with the pairwise post hoc Mann-Whitney U test; *p < 0.05, **p < 0.01, ***p < 0.001

Fig 5: Effects of IL-1β on podoplanin (PDPN), CD106 and NF-κB genes in healthy and diseased tendon cells over time. Tendon cells were derived from healthy hamstring (n = 4 donors) and diseased supraspinatus tendons (n = 4 donors). Cell lysates were sequentially harvested at day 0 (pre-stimulation), day 1 (24 hours after IL-1β treatment, 10 ng/mL-1) and days 2, 3, 4 and 5 when cells were incubated in cytokine-free medium containing vehicle only. mRNA expression is shown for PDPN (a), CD106 (b), IL6 (c), IL8 (d), STAT-1 (e) and IFIT1 (f). Data are mean ± SEM. Statistically significant differences were calculated using the pairwise Mann-Whitney U test for healthy and diseased cells at each time point; *p < 0.05