p ikkα β  (Bioss)

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: Redox Biology

Article Title: Sigma-1 receptor counteracts non-cell-autonomous poly-PR-induced astrocytic oxidative stress in C9orf72 ALS

doi: 10.1016/j.redox.2025.103875

Figure Lengend Snippet: Exogenous poly-PR 20 activates p65 phosphorylation and nuclear translocation in astrocytes via the IKK/IκB pathway. (A) Western blot analysis of phosphorylated p65 (p-p65) and total p65 in astrocytes treated with poly-PR 20 . p65 phosphorylation was notably increased at 3 h post-treatment. (B) Quantification of the p-p65/p65 ratio from three biologically independent experiments, all yielding consistent results. Data are presented as mean ± SEM and analyzed using one-way ANOVA followed by Tukey's multiple comparisons test (∗ p < 0.05). (C) Immunofluorescence staining showing nuclear translocation of p65 in astrocytes treated with FITC-poly-PR 20 for 3 h. (D) Confocal microscopy with Z-stack and line-scan analysis confirmed p65 nuclear localization. Red: p65; Green: FITC-poly-PR 20 ; Blue: DAPI. (E) Semi-quantitative analysis of nuclear versus cytosolic p65 using ImageJ (version 1.53t) from confocal images. Data are presented as mean ± SEM and analyzed using an unpaired two-tailed t -test (∗∗∗ p < 0.001). Total number of cells analyzed: control group, n = 24; FITC-poly-PR 20 group, n = 24. N = 3 biologically independent experiments with consistent results. (F) Western blot analysis showing activation of IKKα/β phosphorylation following poly-PR 20 treatment. (G, H) Quantification of phosphorylated IKKα/β relative to total IKKα (G) and IKKβ (H) revealed a significant increase at 30 min post-treatment. Data are presented as mean ± SEM and analyzed using one-way ANOVA followed by Tukey's multiple comparisons test (∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001). N = 3 biologically independent experiments with consistent results. (I) Western blot analysis showing increased phosphorylation of IκBα in astrocytes following poly-PR 20 treatment. (J) Quantification of the p -IκBα/IκBα ratio showed a significant increase at 2 h. Data are presented as mean ± SEM and analyzed using one-way ANOVA followed by Tukey's multiple comparisons test (∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001). N = 3 biologically independent experiments with consistent results.

Article Snippet: p -IKK α /β , Cell Signaling , 2697s; RRID: AB_2079382.

Techniques: Phospho-proteomics, Translocation Assay, Western Blot, Immunofluorescence, Staining, Confocal Microscopy, Two Tailed Test, Control, Activation Assay