Interleukin-1β affects the phospholipid biosynthesis of fibroblast-like synoviocytes from human osteoarthritic knee joints  K.D. Sluzalska, G. Liebisch, G. Lochnit, B. Ishaque, H. Hackstein, G. Schmitz, M. Rickert, J. Steinmeyer  Osteoarthritis and Cartilage  Volume 25, Issue 11, Pages 1890-1899 (November 2017) DOI: 10.1016/j.joca.2017.07.011 Copyright © 2017 Osteoarthritis Research Society International Terms and Conditions

Fig. 1 Biosynthesis of PLs by cultured human osteoarthritic FLS as modulated by culture conditions and cytokines. A, Lipid composition of FLS obtained from the biosynthesis model after 16 h of labeling. The percentages of PL classes (labeled plus unlabeled PL) of all analyzed PLs (100%) were calculated. Percentages are presented as dot plots with their means ± SDs. Data given above the dot plots depict their corresponding concentrations (nmol/mg protein; n = 6). B and C, Effect of time of labeling on [D9]-choline and [D4]-ethanolamine incorporation into PLs as dependent on the degree of confluency. 100% confluent FLS were labeled with 225 μg/ml of [D9]-choline and 25 μg/ml of [D4]-ethanolamine for 8–36 h, whereas 80% confluent FLS were labeled with 200 μg/ml [D9]-choline and 200 μg/ml of [D4]-ethanolamine for 2–24 h (n = 3). D, Effect of cytokines on the biosynthesis of PLs. FLS were treated with 10 ng/ml IL-1β or IL-6, or 100 ng/ml TNFα for 16 h (n = 6). For each PL class the percentage of stable isotope-labeled PL from total labeled and unlabeled PL (nmol/mg protein) was calculated. Data are presented as means±SDs. * = P ≤ 0.05; ** = P ≤ 0.01; *** = P ≤ 0.001. Osteoarthritis and Cartilage 2017 25, 1890-1899DOI: (10.1016/j.joca.2017.07.011) Copyright © 2017 Osteoarthritis Research Society International Terms and Conditions

Fig. 2 Effect of IL-1β on PL biosynthesis by human osteoarthritic FLS as modulated by inhibitors of cell signaling pathways. The percentages of labeled PL classes from all the corresponding labeled and unlabeled PL class are presented of (A) [D9]-PC, [D4]-PE, [D4]-PE P and (B) [D9]-SM and [D9]-LPC. FLS were treated with 10 ng/ml IL-1β (black bars) alone or with the inhibitors of the cell signaling pathways QNZ (10 μM, NF-κB, grey bars), SB203580 (10 μM, p38, hatched bars) or SP600125 (10 μM, JNK, checked bars) for 16 h (n = 5). Data are presented as means±SDs. * = P ≤ 0.05; ** = P ≤ 0.01; *** = P ≤ 0.001. Osteoarthritis and Cartilage 2017 25, 1890-1899DOI: (10.1016/j.joca.2017.07.011) Copyright © 2017 Osteoarthritis Research Society International Terms and Conditions

Fig. 3 Effect of IL-1β on the biosynthesis of individual PC (A, B) and SM species (C) as modulated by inhibitors of cell signaling pathways. PL biosynthesis was monitored with ESI-MS/MS in the presence of IL-1β (black bars) with or without the addition of the JNK inhibitor SP600125 (hatched bars), the NF-κB activation inhibitor QNZ (white bars) or the p38 MAPK inhibitor SB203580 (grey bars) for 16 h (n = 5). The percentages of stable isotope-labeled PL species were calculated also as a ratio of the corresponding untreated control. As such, data presented are means ± SDs of the x-fold change of % labeled PL species compared to untreated controls (=1). * = P ≤ 0.05; ** = P ≤ 0.01; *** = P ≤ 0.001. Osteoarthritis and Cartilage 2017 25, 1890-1899DOI: (10.1016/j.joca.2017.07.011) Copyright © 2017 Osteoarthritis Research Society International Terms and Conditions

Fig. 4 Effect of IL-1β on the biosynthesis of individual PE P species (A, B) as modulated by inhibitors of cell signaling pathways. PE P biosynthesis was monitored with ESI-MS/MS in the presence of IL-1β (black bars) with or without the addition of the JNK inhibitor SP600125 (hatched bars), NF-κB activation inhibitor QNZ (white bars) or the p38 MAPK inhibitor SB203580 (grey bars) for 16 h (n = 5). The percentages of stable isotope-labeled PE P species were calculated also as a ratio to the corresponding untreated control. As such, data presented are means ± SDs of the x-fold change of % labeled PE P species compared to untreated controls (=1). * = P ≤ 0.05; ** = P ≤ 0.01; *** = P ≤ 0.001. Osteoarthritis and Cartilage 2017 25, 1890-1899DOI: (10.1016/j.joca.2017.07.011) Copyright © 2017 Osteoarthritis Research Society International Terms and Conditions

Fig. 5 Effect of IL-1β on the biosynthesis of individual PE species (A, B) as modulated by inhibitors of cell signaling pathways. PE biosynthesis was monitored with ESI-MS/MS in the presence of IL-1β (black bars) with or without the addition of the JNK inhibitor SP600125 (hatched bars), NF-κB activation inhibitor QNZ (white bars) or the p38 MAPK inhibitor SB203580 (grey bars) for 16 h (n = 5). The percentages of stable isotope-labeled PE species were calculated also as a ratio to the corresponding untreated control. As such, data presented are means ± SDs of the x-fold change of % labeled PE species compared to untreated control (=1). * = P ≤ 0.05, ** = P ≤ 0.01, *** = P ≤ 0.001. Osteoarthritis and Cartilage 2017 25, 1890-1899DOI: (10.1016/j.joca.2017.07.011) Copyright © 2017 Osteoarthritis Research Society International Terms and Conditions