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: Journal of Periodontal & Implant Science

Article Title: Interleukin-6 regulates human ODAM gene expression in gingival epithelial cells

doi: 10.5051/jpis.2402980149

Figure Lengend Snippet: Protein levels were assessed using Western blotting following stimulation with IL-6 (10 ng/mL) for durations of 10 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 6 hours, 12 hours, and 24 hours in Ca9-22 cells. Densitometric analyses of the Western blot results for ODAM, p-STAT3, STAT3, p-p65, p65, p-IKKα/β, IKKβ, and IKKα are presented. The intensity of the α-tubulin band was normalized to 1, and graphs were generated to illustrate the relative intensities of the other protein bands. IL-6: interleukin-6, ODAM: odontogenic ameloblast-associated protein, STAT3: signal transducer and activator of transcription 3, p-STAT3: phosphorylated STAT3, p65: nuclear factor-kappa B p65, p-p65: phosphorylated p65, IKKα: IκB kinase α, IKKβ: IκB kinase β, p-IKKα/β: phosphorylated IκB kinase α/β. The data represent the mean ± standard deviation from 3 independent experiments, with * P <0.05 and ** P <0.01 indicating statistical significance.

Article Snippet: These membranes were incubated with primary antibodies: anti-ODAM (PA5-100656; Invitrogen), anti-signal transducer and activator of transcription (STAT) 3 (10253-2-AP; Proteintech, Rosemont, IL, USA), anti-phospho-STAT3 (Tyr705) (ZRB1006; Sigma-Aldrich, St. Louis, MO, USA), anti-nuclear factor-kappa B (NF-κB) p65 (AF5006; Affinity Biosciences, Cincinnati, OH, USA), anti-phosphorylated (phospho-)NF-κB p65 (Ser536) (AF2006; Affinity Biosciences), anti-IκB kinase α (IKKα) (ab32041; Abcam, Cambridge, UK), anti-IκB kinase β (IKKβ) (#2684; Cell Signaling Technology, Danvers, MA, USA), anti-phospho-IKKα/β (Ser176/180) 16A6 (#2697; Cell Signaling Technology), and anti-α-tubulin (sc-5286; Santa Cruz Biotechnology).

Techniques: Western Blot, Generated, Standard Deviation

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: TANK-binding kinase 1 (TBK1) controls cell survival through PAI-2/serpinB2 and transglutaminase 2.

doi: 10.1073/pnas.1119296109

Figure Lengend Snippet: Fig. 1. TBK1 protects MEFs from TNF-induced apoptosis and modulates IKK-dependent phosphorylation of RelA/p65. (A) Wt and Tbk1−/−MEFs were treated for 24 h with TNF (25 ng/mL) alone or for 3 h with TNF in the presence of CHX (10 μg/mL). Apoptotic cells were detected by TUNEL assays. The bars represent averages ± SD of three different experiments. Approximately 1,200 cells were counted in each experiment. (B) MEFs treated with TNF and CHX for the indicated times were analyzed for caspase-8 and caspase-3 activation by immunodetection of their cleaved forms. (C) Cell extracts prepared from Tbk1−/−

Article Snippet: Antibodies against RelA/p65 (S536) (no. 3031), phospho-IκBα (no. 9241), phospho-MAPKs (no. 9910), phospho-cJun (no. 9261), ERK, p38 (no. 9212), TBK1 (no. 3012), Bcl-XL (no. 2764), c-IAP1 (no. 4952), mBID (no. 2003), caspase-3 (no. 9662), cleaved caspase-3 (no. 9661), and cleaved PARP (no. 9544) were from Cell Signaling; antibodies to IKKγ (no. 557383), JNK1 (no. 551196), XIAP (no. 610716), CD95/Fas receptor (554254), caspase-8 (no. 559932), and procaspase-3 (no. 65906E) were from BD Transduction Laboratories; antibodies to IKKα (no. IMG-136A), IKKβ (no. IMG-129A), TBK1 (IMG-139A), and IκBα (no. IMG-127A) were from Imgenex; antibodies against RelA (sc-372 and sc-372G), RelB, cRel, IκBβ, HSP60, and PAI-2 (sc-25746) were from Santa Cruz Biotechnology; anti-p65/RelA (CT), anti–phospho-ATF-2 (no. 05–891), anti-TG2 (no. 06–471), and anti-caspase-1 (no. 06–503) were from Upstate Biotechnology; anti–CREB-1 (AB3006) was from Millipore; anti-FLIPα (CT; no. 1161) was from ProSci, anti-TG2 (AB-4) was from Neomarkers (Lab Vision), anti-HA (clone 3F10) was from Roche, anti-cAIP2 was from R&D Systems, and anti-CD31 was from Abcam.

Techniques: Phospho-proteomics, TUNEL Assay, Activation Assay, Immunodetection

Journal: bioRxiv

Article Title: A genome-wide CRISPR screen in human prostate cancer cells reveals drivers of macrophage-mediated cell killing and positions AR as a tumor-intrinsic immunomodulator

doi: 10.1101/2023.06.06.543873

Figure Lengend Snippet: A. Schematic overview of IKK signaling. Canonical NF-κB is regulated by the IKK complex, which consists of the IKKα, β, and γ subunits. Phosphorylation of IκBα leads to its degradation, allowing the p65-p50 dimer to accumulate in the nucleus and regulate transcription of target genes. B. Relative mRNA levels of IKBKB, IKBKG and CHUK (normalized to TBP ) in IKBKB-KO , IKBKG-KO and CHUK-KO LNCaP cells (n=3, mean values are depicted and error bars represent the standard deviation). P-value was calculated using a two-way ANOVA test. C. Protein levels of IKBKB, IKBKG and CHUK protein levels in IKBKB-KO , IKBKG-KO and CHUK-KO LNCaP cells. Actin and HSP90 are used as loading control. Cas9 was used as a control for efficient lentiCRISPRv2 transfection. D. Crystal violet assay of NT control and IKBKB-KO , IKBKG-KO and CHUK-KO cells cultured as a monoculture (up) and co-culture (down) with macrophages. Cells were cultured for 3 days. Crystal violet staining was quantified using Fiji software.

Article Snippet: After transfer, membranes were stained with Ponceau S and subsequently blocked in 4% BSA (A8022, Sigma/Merck) in 1x PBS-Tween (137 mM NACl, 10 mM Na 2 HP04, 1.5 mM KH 2 PO 4 , 2.6 mM KCl, 0.1% Tween-20) for 1h and incubated with primary antibodies against IKKα (2682, Cell signaling, 1:1000), IKKβ (10AG2, Novus Biological, 1:000), IKKγ (2685, Cell signaling, 1:1000), PKCδ (2058, Cell signaling, 1:1000), Actin (MAB1501R, Merck, 1:1000), HSP90 (sc-12119, Santa Cruz Biotechnologies, 1:1000) diluted in 4% BSA/PBS-T. Blots were incubated overnight at 4°C and washed five times with PBS-T prior to incubation with secondary antibodies donkey-α-mouse 680 RD (926-68073, LI-COR Biosciences, 1:10000) and donkey-α-rabbit 800 CW (926-32213, LI-COR Biosciences, 1:10000), diluted in 4% BSA/PBS-T for 1h.

Techniques: Phospho-proteomics, Standard Deviation, Control, Transfection, Crystal Violet Assay, Cell Culture, Co-Culture Assay, Staining, Software