Journal: Scientific Reports
Article Title: Catabolic mediators from TLR2-mediated proteoglycan aggrecan peptide-stimulated chondrocytes are reduced by Lactobacillus -conditioned media
doi: 10.1038/s41598-024-68404-9
Figure Lengend Snippet: Effects of TLR2 blockade and Lactobacillus- conditioned media treatment on intracellular signaling activation in PG peptide-stimulated chondrocytes. Chondrocytes isolated from OA patients (N = 5) were treated with 20 μg/ml of anti-TLR2 antibodies or 10% of LCM, L. salivarius -B60 for 1 h before stimulating with IFNγ and PG peptides (p16-31, p263-280 and p2379-2394) or control peptides (32-mer and Col2 peptides). Intracellular phosphorylated proteins; p-STAT3, p-IkBα, p-ERK1/2, p-p38 and p-MAPK9 (JNK2) were determined by flow cytometry. ( A ) Bar graph showing the fold change of mean fluorescent intensity of phosphorylated proteins of stimulated chondrocyte which was divided by that of unstimulated chondrocytes. Statistical significance was calculated by one-way ANOVA with post-hoc Turky HSD. ( B ) Comparison of mean fluorescence intensity (MFI) of phosphorylated protein expression after stimulating with IFNγ and PG peptides between no blocking and anti-TLR2 antibody treatment (upper panel) or between no blocking and L. salivarius -B60 LCM treatment in each individual. Statistical significance was calculated by paired-T test. ( C ) Bar graphs showing the comparison of percentage of phosphorylated protein- expressing chondrocytes in no blocking, anti-TLR2 antibody treatment and L. salivarius -B60 LCM treatment conditions after stimulation with IFNγ and PG peptides or control peptides. Data was shown as mean ± SEM and statistical significance was calculated by two-way ANOVA with Bonferroni post hoc test (*, p < 0.0500; **, p < 0.0100; ***, p < 0.0010; ****, p < 0.0001).
Article Snippet: Intracellular signaling proteins were labeled with phospho-specific antibodies; anti-human phospho-p38 (Thr180/Try182)-PE/Cy7 (Clone 4NIT4KK, Invitrogen), anti-human phospho-MAPK9 (pJNK2) (Thr183/Tyr185)-APC (Clone SAPKT183Y185-A11, Invitrogen), anti-human phospho-ERK1/2 (Thr202/Try204)-PE/Cy5 (Clone 6B8B69, Biolegend), anti-human phospho-STAT3 (Tyr705)-AF488 (Clone 13A3-1, Biolegend) and anti-human phospho-IkBα (Ser32/Ser36)-PE (Clone RILYB3R, Invitrogen) antibodies for 1 h at room temperature.
Techniques: Activation Assay, Isolation, Control, Flow Cytometry, Comparison, Fluorescence, Expressing, Blocking Assay
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