Journal: Bone & Joint Research
Article Title: CXCL5 suppresses osteoclastogenesis and protects against lipoteichoic acid-induced bone loss by modulating PLCγ2 and c-Fos signalling in gram-positive periprosthetic joint infection
doi: 10.1302/2046-3758.153.BJR-2025-0290.R1
Figure Lengend Snippet: Chemokine (C-X-C motif) ligand 5 (CXCL5) inhibits receptor activator of nuclear factor kappa-B ligand (RANKL)-induced osteoclast differentiation. a), c), and e) Immunofluorescence staining confirmed that CXCL5 (1 or 50 ng/ml) inhibits RANKL-induced differentiation of RAW264.7 cells into cathepsin K (CTSK)-positive osteoclast-like cells. b), d), and f) CXCL5 decreased the differentiation of RAW264.7 cells into tartrate-resistant acid phosphatase (TRAP)-positive osteoclast-like cells. Purple indicates TRAP expression, and cyan indicates nuclei. g) and h) Western blot analysis showed that CXCL5 (1 ng/ml) inhibited RANKL-induced differentiation of RAW264.7 cells by suppressing the transcription factor nuclear factor of activated T-cells cytoplasmic 1 (NFATc1), resulting in decreased CTSK expression at 72 hours of treatment. Data are presented as the mean (standard error of the mean (SEM)) and analyzed using one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test. *p < 0.05, **p < 0.01, ***p < 0.001. DAPI, 4′,6-diamidino-2-phenylindole.
Article Snippet: Western blotting was performed using primary antibodies against the following targets: nuclear factor of activated T-cells cytoplasmic 1 (NFATc1) (4389), phospho-phospholipase Cγ2 (PLCγ2) (Tyr1217, 3871T), PLCγ2 (3872T), phospho-PLCγ1 (Tyr783, 2821T), PLCγ1 (5690T), c-Fos (4384), calcineurin A (2614S), phospho-p38 mitogen-activated protein kinase (MAPK) (Thr180/Tyr182, 9211), p38 MAPK (8690), phospho-SAPK/JNK (Thr183/Tyr185, 4668), SAPK/JNK (9252), phospho-ERK1/2 (4370), ERK1/2 (9102), phospho-nuclear factor-kappa B (NF-κB) p65 (Ser536, 3033), NF-κB p65 (8242), phospho-signal transducer and activator of transcription 5 (STAT5) (Tyr694, 9359S), STAT5 (94205T), phospho-STAT6 (Tyr641, 56554S), STAT6 (5397S), phospho-FAK (Tyr397, 3283S), and FAK (3285T) (all from Cell Signaling Technology, USA); TRAP (ab191406) and CXCR2 (ab217314; Abcam); CTSK (sc-48353), tumour necrosis factor receptor-associated factor 6 (TRAF6; sc-8409), and β-actin (sc-47778; Santa Cruz Biotechnology, USA).
Techniques: Immunofluorescence, Staining, Expressing, Western Blot, Comparison