Fig 2: (A) Representative images of HSE cell line after 24 h stimulation (630× magnification, 1 µg/mL NP, 10 ng/mL TNFα or the combination), immunolabeled with C5aR1 and CD55 specific antibodies and negative control of the staining. Green (Alexa 488) = C5aR1, red (Cy3) = CD55, blue (DAPI) = cell nuclei. Scale bar = 25 µm. Graphic representation of relative (B) C5aR1 and (C) CD55 protein fluorescence intensity, n = 4, mean with standard deviation (SD). Control has been normalized to 100. One sample t-test with significance in relation to control. Repeated Measures one-way ANOVA using Tukey’s multiple comparisons.

Fig 3: Graphic representation of relative gene expression of complement factors (C5aR1, CD46, CD55 and CD59) and cytokines (TNFα and IL-6) in non-stimulated synoviocyte cell lines K4IM and HSE (A) as well as K4IM (B) or HSE (C) when stimulated separately for 24 h with 1 µg/mL NP, 10 ng/mL TNFα or the combination of both. n = 3–5. Mean with standard deviation (SD). Control has been normalized to 100. One sample t-test with significance in relation to control (*). Repeated Measures one-way ANOVA using Tukey’s multiple comparisons. * = p ≤ 0.05, ** = p ≤ 0.01, **** = p ≤ 0.001.

Fig 4: (A) Representative images of K4IM cell line after 24 h stimulation (630× magnification, 1 µg/mL NP, 10 ng/mL TNFα or the combination), immunolabeled with C5aR1 and CD55 specific antibodies and negative control of the staining. Green (Alexa 488) = C5aR1, red (Cy3) = CD55, blue (DAPI) = cell nuclei. Scale bar = 25 µm. Graphic representation of relative (B) C5aR1 and (C) CD55 protein fluorescence intensity, n = 4, mean with standard deviation (SD). Control has been normalized to 100. One sample t-test with significance in relation to control. Repeated Measures one-way ANOVA using Tukey’s multiple comparisons.

Fig 5: Graphic representation of relative chondrocytes gene expression of C5aR1 (A), C5 (B), and CD59 (C) after 24 h and 72 h of stimulation. n = 3 (24 h stimulation) and n = 5 (72 h stimulation) independent experiments with chondrocytes from different donors. Mean with standard deviation. Control has been normalized to 1. One sample t-test with significance in relation to control (*). Mixed-effects analysis using post-hoc Tukey’s multiple comparisons (#). */# = p ≤ 0.05; ** = p ≤ 0.01, *** = p ≤ 0.001.