Journal: Oncology reports

Article Title: Hsp90 inhibitor induces KG-1a cell differentiation and apoptosis via Akt/NF-κB signaling.

doi: 10.3892/or.2017.5797

Figure Lengend Snippet: Figure 4. SNX-2112 inhibits Akt and NF-κB signaling. (A) KG-1a cells were treated with indicated concentrations of SNX-2112 for 48 h. Hsp90, IKKα, IKKβ, Akt and p-Akt were analyzed by western blotting relative to that of GADPH. (B) KG-1a cells were treated with an NF-κB pathway inhibitor (Bar, 0.5 µM) or left untreated (control) for 48 h, and cell proliferation was assessed with CCK-8 assay. (C and D) KG-1a cells were treated with Bar (0.5 µM) or a vehicle for 48 h and Annexin V/PI staining was detected after 48 h by flow cytometry. Data are presented as the mean ± SEM (n=3/group); ****P<0.0001 compared to the corresponding control.

Article Snippet: Antibodies against glyceraldehyde 3-phosphate dehydrogenase (GAPDH), caspase-3, Bcl-xl, Bcl-2, Akt, p-Akt (Thr308), IKKα, IKKβ, p65, p-p65, IκB, and PU.1 were purchased from Cell Signaling Technology (Beverly, MA, USA), and cluster of differentiation (CD)11b was purchased from BD Biosciences (Franklin Lakes, NJ, USA).

Techniques: Western Blot, Control, CCK-8 Assay, Staining, Flow Cytometry

Journal: Oncology reports

Article Title: Hsp90 inhibitor induces KG-1a cell differentiation and apoptosis via Akt/NF-κB signaling.

doi: 10.3892/or.2017.5797

Figure Lengend Snippet: Figure 4. Continued. SNX-2112 inhibits Akt and NF-κB signaling. (E) KG-1a cells were treated with Bar (0.5 µM) or left untreated (control) for 48 h. IKKα, IKKβ and p65 levels were analyzed by western blotting relative to that of GADPH. (F) KG-1a cells were treated with indicated concentrations of SNX-2112 for 48 h. IκB, p65 and p-p65 levels were analyzed by western blotting relative to that of GADPH. (G) KG-1a cells were treated with indicated concentrations of SNX-2112 for 48 h and p65 expression was detected by immunofluorescence labeling. Nuclear cytoplasmic expression of NF-κB p65 was decreased by SNX-2112 treatment.

Article Snippet: Antibodies against glyceraldehyde 3-phosphate dehydrogenase (GAPDH), caspase-3, Bcl-xl, Bcl-2, Akt, p-Akt (Thr308), IKKα, IKKβ, p65, p-p65, IκB, and PU.1 were purchased from Cell Signaling Technology (Beverly, MA, USA), and cluster of differentiation (CD)11b was purchased from BD Biosciences (Franklin Lakes, NJ, USA).

Techniques: Control, Western Blot, Expressing, Immunofluorescence, Labeling

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: 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