Journal: International Journal of Medical Sciences

Article Title: Artemisia argyi -enhanced Mesenchymal Stem Cell Exosomes Alleviates Inflammation in C28/I2 Chondrocytes by inhibiting NF-κB

doi: 10.7150/ijms.126119

Figure Lengend Snippet: C28/I2 cells were exposed to H₂O₂ (100 μM, 2 h) followed by treatment with AA-enhanced WJSC-conditioned medium or exosomes. Cell viability, exosome uptake, NF-κB signaling proteins, cartilage-associated markers, and intracellular ROS levels were analyzed. (A) AA-enhanced conditioned medium at concentrations of 1%, 3%, 5%, 7%, and 9% for 24 hours significantly promoted C28/I2 cell proliferation compared to the control group. (B) After 2 hours of oxidative stress with H₂O₂, treatment with AA-enhanced conditioned medium restored cell viability in a dose-dependent manner. (C) Similarly, treatment with increasing concentrations of AA-enhanced WJSCs exosomes after H₂O₂ exposure enhanced cell viability. (D) Fluorescence microscopy confirmed cellular uptake of PKH67-labeled exosomes, with F-actin filaments stained in red (Scale bar = 20 μm). (E) Immunoblot analysis showed decreased expression of inflammation markers (p-IKKα/β, p-NF-κB, IκBα) and OA marker MMP13, with increased COL2A1 at higher concentrations of AA-enhanced conditioned medium (5%, 7%, 9%). (F) Similarly, AA-enhanced WJSCs exosomes (80 μg/mL) modulated inflammatory and OA markers. (G) mRNA expression of ADAMTS14, ADAMTS15, TNF-α, and IL-17A was normalized to β-actin using the 2-∆∆Ct method. (H) ROS generation was assessed by DCFDA staining. Fluorescence microscopy showed reduced ROS levels in cells treated with AA-enhanced exosomes (80 μg/mL) compared to the H₂O₂ group (Scale bar = 40 μm). (I) Quantification confirmed significant reduction in ROS levels, emphasizing the antioxidant effects of AA- conditioned vesicles. Data are presented as mean ± SD (n = 3), * p < 0.05, ** p < 0.01, *** p < 0.001, ****p < 0.0001 vs. untreated control group; # p < 0.05, ## p < 0.01, ### p < 0.001, #### p < 0.0001 vs. H₂O₂ treated group.

Article Snippet: The following antibodies were used in the current study: CD9 (#13174; Cell signaling technology, Danvers, MA, USA), CD63(Merck Millipore; Burlington, MA, USA), CD81(sc-166028; Santa Cruz Biotechnology, Dallas, TX, USA), Calnexin (#2679, Cell signaling), β-actin(sc-47778, Santa Cruz), p-IKKα/β (bs-3237R; Bioss Antibodies, Woburn, MA, USA), p-NF-κB p-p65 (#3033, Cell signaling), NF-κB p65(#8242, Cell signaling), p-IκB-α (#2859, Cell signaling), IκB-α (sc-1643, Santa Cruz), COL2A1 (GB11021; Servicebio, Wuhan, Hubei, China), MMP-13 (GTX100665; GeneTex, Irvine, CA, USA), Nanog (#4903, Cell Signaling), KLF4 (#4038, Cell signaling), CXCR4 (60042-1-Ig; Proteintech, San Diego, CA, USA), CXCR7 (bs-4897R, Bioss), CD44 (#3570, Cell signaling), CD90 (sc-53456, Santa Cruz).

Techniques: Control, Fluorescence, Microscopy, Labeling, Staining, Western Blot, Expressing, Marker

Journal: International Journal of Medical Sciences

Article Title: Artemisia argyi -enhanced Mesenchymal Stem Cell Exosomes Alleviates Inflammation in C28/I2 Chondrocytes by inhibiting NF-κB

doi: 10.7150/ijms.126119

Figure Lengend Snippet: Verification of AA-enhanced exosomes reducing H₂O₂-induced inflammation in C28/I2 cells and supporting cartilage homeostasis via inhibition of the NF-κB pathway using an NF-κB activator. C28/I2 cells were pretreated with H 2 O 2 for 2 hours and then co-treated with standard WJSCs exosomes or AA-enhanced WJSCs exosomes (80 μg/mL) with NF-κB activator (5μM) for 22 hours. (A) Western blot analysis reveals expression levels of key inflammation-related proteins, including p-IKKα/β, p-NF-κB, and IκBα, as well as OA markers MMP13 and COL2A1. (B) Translocation of p65 was determined using a NF-κB p65 antibody and an Alexa Fluor 488-conjugated anti-rabbit IgG antibody. Nuclei were counterstained with DAPI. Scale bar = 40 μm.

Article Snippet: The following antibodies were used in the current study: CD9 (#13174; Cell signaling technology, Danvers, MA, USA), CD63(Merck Millipore; Burlington, MA, USA), CD81(sc-166028; Santa Cruz Biotechnology, Dallas, TX, USA), Calnexin (#2679, Cell signaling), β-actin(sc-47778, Santa Cruz), p-IKKα/β (bs-3237R; Bioss Antibodies, Woburn, MA, USA), p-NF-κB p-p65 (#3033, Cell signaling), NF-κB p65(#8242, Cell signaling), p-IκB-α (#2859, Cell signaling), IκB-α (sc-1643, Santa Cruz), COL2A1 (GB11021; Servicebio, Wuhan, Hubei, China), MMP-13 (GTX100665; GeneTex, Irvine, CA, USA), Nanog (#4903, Cell Signaling), KLF4 (#4038, Cell signaling), CXCR4 (60042-1-Ig; Proteintech, San Diego, CA, USA), CXCR7 (bs-4897R, Bioss), CD44 (#3570, Cell signaling), CD90 (sc-53456, Santa Cruz).

Techniques: Inhibition, Western Blot, Expressing, Translocation Assay

Journal: Cell Communication and Signaling : CCS

Article Title: Unanchored K63-linked polyubiquitin chains: a novel second messenger involved in G protein-coupled receptors early signaling events

doi: 10.1186/s12964-025-02646-6

Figure Lengend Snippet: TRAF6 rapidly produces free K63-linked polyubiquitin chains, which interact with the IKK complex in response to GPCR stimulation. A Wild-type and TRAF6 knockout MEFs were serum-starved for 5 h and then exposed to Thr (1 U/mL) for 10 min. WCE were prepared and subjected to immunoprecipitation using anti-IKKγ antibody before immunoblotting analysis using the indicated antibodies. Data are representative of two independent experiments. B to D VSMC were serum-starved for 24 h and then exposed to Thr (1 U/mL), LPA (10 μM) and Ang II (100 nM) for the indicated times. WCE were prepared and subjected to immunoprecipitation using anti-IKKγ antibody. Immunoprecipitated proteins were treated with or without isoT (200 nM) before immunoblotting analysis using the indicated antibodies. Data are representative of a minimum of 2 independent experiments. E ) HASMC were serum-starved for 24 h and then exposed to Ang II (1 μM) for the indicated times. WCE were prepared and subjected to immunoprecipitation using anti-IKKβ antibody. Immunoprecipitated proteins were treated with or without isoT (200 nM) before immunoblotting analysis using the indicated antibodies. The data are representative of two independent experiments

Article Snippet: Commercial antibodies were purchased from the following suppliers: anti-TRAF6 (IMG-5654–2) 1μg/mL was from Imgenex; anti-phospho-p38 MAP Kinase Thr180/Tyr182 (#9211) 1: 1000, anti-phospho-SAPK/JNK Thr183/Tyr185 (#9251) 1: 1000, anti-phospho-TAK1 Thr187 (#4536) 1: 1000, anti-phospho-IkBα Ser32/Ser36 clone 5A5 (#9246) 1: 1000, anti-IKKβ clone D30C6 (#8943) 1: 1000, anti-p38 MAPK (#9202) 1: 1000, anti-SAPK/JNK (#9252) 1: 1000, anti-TAK1 (#4505) 1: 1000, anti-IkBα (#9242) 1: 1000 and anti-NF-κB p65 (#8242) 1μg/mL (immunofluorescence (IF) studies) were from Cell Signaling Technology; anti-IKKγ FL-149 (sc-8330) 1μg/mL and anti-TAB2 clone H-300 (sc-20756) 1μg/mL (Western blot studies) or 2 μg/mL confocal microscopy (CF) were from Santa Cruz Biotechnology; anti-Human Ub-K63 clone HWA4C4 (#05–1313) 2 μg/mL was from Millipore; phospho-IKKα/β Ser180/Ser181 (SAB4301429) 1: 1000, anti-Flag (F7425) 1μg/mL (Western blot studies), anti-Flag M2 (F3165) 1μg/mL (CF), anti-TUBA4A (T6199) 1μg/mL (IF) and anti-β-actin clone AC-74 (A5316) 1: 10 000 were from Sigma; anti-IKKβ (A301-827A) and anti-TAK1 (A301-916A) used explicitly for immunoprecipitation were from Bethyl Laboratories; anti-glutathione-S-transferase (GST) clone 8-326 (AB_10979611) was from ThermoFisher Scientific.

Techniques: Knock-Out, Immunoprecipitation, Western Blot