Journal: Cell Death & Disease

Article Title: Mesenchymal stem cells reverse EMT process through blocking the activation of NF-κB and Hedgehog pathways in LPS-induced acute lung injury

doi: 10.1038/s41419-020-03034-3

Figure Lengend Snippet: a The cocultured system with LPS-treated MLE-12 cells and MSCs was built. b Luciferase reporter assays detected the relative luciferase activity of various signaling pathways (Notch, Wnt, Nanog, NF-κB, PI3K/AKT, Oct4, Hedgehog, MAPK/JNK, and MAPK/ERK) in LPS-treated MLE-12 cells treated with or without MSC. c The mRNA level of Ikbkb, Chuk, Ikbkg, or RelA was examined by RT-qPCR after cocultured with MSC. d Western blot analysis determined the levels of IKKβ, p-IκBα, and p-IκBβ in the whole cell lysates as well as the nuclear protein level of p65 after cocultured with MSCs. e RT-qPCR analyzed the mRNA levels of hedgehog pathway key factors (Shh, Dhh, Ihh, Ptch1, Smo, and Gli1) in control cells and cocultured cells. f The luciferase activity of hedgehog pathway was detected in cocultured cells. g Relative mRNA level of Shh was measured by RT-qPCR in LPS-treated MLE-12 cells under four different conditions (control, MSC, KINK-1, and KINK-1+MSC). h Relative luciferase activity of hedgehog pathway was measured in LPS-treated MLE-12 cells under the same four conditions. ** p < 0.01. n.s. no statistical significance.

Article Snippet: The kinase inhibitor of NF-κB-1 (KINK-1; 5 μM), IKKβ inhibitor, was procured from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Luciferase, Activity Assay, Protein-Protein interactions, Quantitative RT-PCR, Western Blot, Control

Journal: Cell Death & Disease

Article Title: Mesenchymal stem cells reverse EMT process through blocking the activation of NF-κB and Hedgehog pathways in LPS-induced acute lung injury

doi: 10.1038/s41419-020-03034-3

Figure Lengend Snippet: a After treated with CHX in three different time points, the protein level of IKKβ was tested in LPS-treated MLE-12 cells with or without MSC-exosome. b The protein level of IKKβ was detected in LPS-treated MLE-12 cells treated with control or MSC-exosome or control+MG132 or MSC-exosome+MG132. c Ubiquitination assay detected the ubiquitination of IKKβ protein in LPS-treated MLE-12 cells treated with MSC-exosome. d Pull-down silver staining was applied to unveil the proteins that might interact with IKKβ. e Co-IP assay demonstrated the interaction between Usp5 and IKKβ. f RT-qPCR and western blot examined the overexpression efficiency of Usp5 and the protein level of IKKβ in response to Usp5 overexpression. g Western blot analyzed the protein level of IKKβ in LPS-treated MLE-12 cells under four situations (control, MSC-exosome, MSC-exosome+pcDNA3.1/Usp5, or MSC-exosome+pcDNA3.1/Usp5+miR-182-5p inhibitor). h Luciferase reporter assays indicated the relative luciferase activity of Usp5 promoter in LPS-treated MLE-12 cells treated with MSC-exosome. i RT-qPCR and western blot detected the mRNA level and protein level of Usp5 in LPS-treated MLE-12 cells treated with MSC-exosome or MSC/sh-Dicer-exosome. ** p < 0.01. n.s. no statistical significance.

Article Snippet: The kinase inhibitor of NF-κB-1 (KINK-1; 5 μM), IKKβ inhibitor, was procured from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Control, Ubiquitin Proteomics, Silver Staining, Co-Immunoprecipitation Assay, Quantitative RT-PCR, Western Blot, Over Expression, Luciferase, Activity Assay

Journal: Cell Death & Disease

Article Title: Mesenchymal stem cells reverse EMT process through blocking the activation of NF-κB and Hedgehog pathways in LPS-induced acute lung injury

doi: 10.1038/s41419-020-03034-3

Figure Lengend Snippet: a StarBase v2.0 predicted miRNAs targeted to Usp5 were subjected to RT-qPCR analysis in LPS-treated MLE-12 cells with or without MSC coculture. b RT-qPCR analyzed miR-23a-3p expression in LPS-treated MLE-12 cells treated with MSC-exosome. c RT-qPCR analyzed miR-23a-3p expression in LPS-treated MLE-12 cells transfected with miR-23a-3p mimics. d The binding sequence between miR-23a-3p and Usp5 was shown. e Luciferase reporter assay examined the luciferase activity of indicated vectors in LPS-treated MLE-12 cells and HEK-293T cells co-transfected with miR-23a-3p mimics or NC mimics. f The expression level of Usp5 was detected by RT-qPCR in LPS-treated MLE-12 cells after the transfection of miR-23a-3p mimics. g Western blot measured the protein level of IKKβ in LPS-treated MLE-12 cells treated with different groups (control, MSC-exosome, MSC-exosome+miR-23a-3p inhibitor or MSC-exosome+miR-23a-3p inhibitor+miR-182-5p inhibitor). h After CHX treatment, the half-life of IKKβ was detected in LPS-treated MLE-12 cells transfected with NC mimics, miR-23a-3p mimics or miR-23a-3p mimics+pcDNA3.1/Usp5. i The ubiquitination level of IKKβ was detected in LPS-treated MLE-12 cells transfected with NC mimics, miR-23a-3p mimics or miR-23a-3p mimics+pcDNA3.1/Usp5. j Western blot examined the protein level of IKKβ in LPS-treated MLE-12 cells under seven conditions (control, MSC-exosome, MSC-exosome+miR-23a-3p inhibitor, MSC-exosome+miR-182-5p inhibitor, control+MG132, MSC-exosome+MG132, and MSC-exosome+miR-23a-3p inhibitor+miR-182-5p inhibitor+MG132). ** p < 0.01. n.s. no statistical significance.

Article Snippet: The kinase inhibitor of NF-κB-1 (KINK-1; 5 μM), IKKβ inhibitor, was procured from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Quantitative RT-PCR, Expressing, Transfection, Binding Assay, Sequencing, Luciferase, Reporter Assay, Activity Assay, Western Blot, Control, Ubiquitin Proteomics

Journal: Cell Death & Disease

Article Title: Mesenchymal stem cells reverse EMT process through blocking the activation of NF-κB and Hedgehog pathways in LPS-induced acute lung injury

doi: 10.1038/s41419-020-03034-3

Figure Lengend Snippet: Rescue assays were carried out in LPS-treated MLE-12 cells under four different contexts (control, MSC-exosome, MSC-exosome+miR-182-5p inhibitor, and MSC-exosome+miR-182-5p inhibitor+miR-23a-3p inhibitor). a The levels of nuclear p65, IKKβ, p-IKBα, and p-IKBβ were detected in LPS-treated MLE-12 cells using western blot. b IF staining examined the nuclear translocation of p65 in LPS-treated MLE-12 cells under diverse conditions. Scale bar = 50 μm. c The protein level of p65 in nucleus or cytoplasm was examined by western blot analysis in LPS-treated MLE-12 cells with MSC-exosome, MSC-exosome+miR-182-5p inhibitor or MSC-exosome+miR-23a-3p inhibitor. d Luciferase reporter assay examined the luciferase activity of hedgehog pathway in LPS-treated MLE-12 cells. e – i RT-qPCR detected the mRNA level of E-cadherin, α-SMA, TGF-β1, Collagen type I, and Collagen type III in indicated LPS-treated MLE-12 cells. j Western blot detected the protein level of E-cadherin, α-SMA, TGF-β1, Collagen type I, and Collagen type III in indicated LPS-treated MLE-12 cells. * p < 0.05, ** p < 0.01.

Article Snippet: The kinase inhibitor of NF-κB-1 (KINK-1; 5 μM), IKKβ inhibitor, was procured from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Control, Western Blot, Staining, Translocation Assay, Luciferase, Reporter Assay, Activity Assay, Quantitative RT-PCR

Journal: Cardiovascular Research

Article Title: Ex vivo Ikkβ ablation rescues the immunopotency of mesenchymal stromal cells from diabetics with advanced atherosclerosis

doi: 10.1093/cvr/cvaa118

Figure Lengend Snippet: Advanced glycolytic end (AGE) products activates IKKβ signalling cascade and reduce Mesenchymal stromal cells (MSCs) immunopotency. (A) Western-blot analysis representative of two independent experiments with similar results showing the activation of the IRAK4–TAK1–IKKβ–p65/NF-κB signalling cascade on healthy MSCs that are exposed to 5, 10, and 15 µg/mL AGE products for 24 h. (B–D) AGE induced an increase in the production of IKKβ–NF-κB-regulated pro-inflammatory cytokine IL-6 (***P = 0.0002, n = 8), and chemokines IL-8/CXCL8 (***P = 0.0006, n = 8), MCP-1/CCL2 (***P = 0.0002, n = 8). (E) Reduced immunosuppressive function of MSCs after AGE products (***P = 0.0002, n = 8). Each dot represents different biological replicates (i.e. single healthy control MSCs). Mann–Whitney U test was used to compare two independent groups. Abbreviations: MSCs, mesenchymal stromal cells; TAK-1, transforming growth factor‐β‐activating kinase1; IKKβ, inhibitor of nuclear factor kappa-B kinase subunit beta; p65, nuclear factor kappa-light-chain-enhancer of activated B cells RelA subunit; IRAK4, interleukin-1 receptor-associated kinase 4; IL-6, interleukin-6; IL-8, interleukin-8; MCP-1, monocyte chemoattractant protein-1; PBMCs, peripheric blood mononuclear cells.

Article Snippet: Six different human IKKβ guide RNAs (gRNAs) (see Supplementary material online , Table S1 ) and non-targeting gRNAs were purchased from GenScript (NJ, USA) and sub-cloned in pLentiCRISPR v2 using BsmBI restriction site (Addgene Plasmid No. 52961).

Techniques: Western Blot, Activation Assay, Control, MANN-WHITNEY

Journal: Cardiovascular Research

Article Title: Ex vivo Ikkβ ablation rescues the immunopotency of mesenchymal stromal cells from diabetics with advanced atherosclerosis

doi: 10.1093/cvr/cvaa118

Figure Lengend Snippet: Constitutively activated forms of inflammation-activated protein kinases and increased pro-inflammatory cytokine secretion in atherosclerosis+T2DM MSCs. Augmented active T-loop phosphorylated forms of (A) IRAK 4 (*P = 0.05, atherosclerosis MSCs n = 5, atherosclerosis+T2DM MSCs n = 6), (B) TAK1 (*P = 0.02, atherosclerosis MSCs n = 10, atherosclerosis+T2DM MSCs n = 12) and (C) IKKβ (****P < 0.000, atherosclerosis MSCs n = 10, atherosclerosis+T2DM MSCs n = 11); (D) Increased phosphorylation of p65 NF-κB transcription factor on Ser536 located in its transactivation domain (*P = 0.05, atherosclerosis MSCs n = 10, atherosclerosis+T2DM MSCs n = 12) and (E–G) Increased production of IKKβ–NF-κB-dependent pro-inflammatory cytokine IL-6 (***P = 0.0002, atherosclerosis MSCs n = 12, atherosclerosis+T2DM MSCs n = 11), and chemokines IL-8/CXCL8 (***P = 0.0006, atherosclerosis MSCs n = 11, atherosclerosis+T2DM MSCs n = 10), MCP-1/CCL2 (***P = 0.0002, atherosclerosis MSCs n = 11, atherosclerosis+T2DM MSCs n = 11). Each dot represents different biological samples (i.e. MSC from different donors). Mann–Whitney U test was used to compare two independent groups. Abbreviations: T2DM, type 2 diabetes mellitus; MSCs, mesenchymal stromal cells; TAK-1, transforming growth factor‐β‐activating kinase1; IKKβ, inhibitor of nuclear factor kappa-B kinase subunit beta; p65, nuclear factor kappa-light-chain-enhancer of activated B cells RelA subunit; IRAK4, interleukin-1 receptor-associated kinase 4; IL-6, interleukin-6; IL-8, interleukin-8; MCP-1, monocyte chemoattractant protein-1.

Article Snippet: Six different human IKKβ guide RNAs (gRNAs) (see Supplementary material online , Table S1 ) and non-targeting gRNAs were purchased from GenScript (NJ, USA) and sub-cloned in pLentiCRISPR v2 using BsmBI restriction site (Addgene Plasmid No. 52961).

Techniques: Phospho-proteomics, MANN-WHITNEY

Journal: Cardiovascular Research

Article Title: Ex vivo Ikkβ ablation rescues the immunopotency of mesenchymal stromal cells from diabetics with advanced atherosclerosis

doi: 10.1093/cvr/cvaa118

Figure Lengend Snippet: Pharmacological inhibition of IKKβ reduces pro-inflammatory cytokine and chemokine secretion and rescues in vitro immunopotency of atherosclerosis+T2DM MSCs. (A–C) MLN120B treatment reduces the secretion of IL-6 (***P = 0.001, n = 11), IL-8/CXCL8 (**P = 0.003, n = 9), and MCP-1/CCL2(**P = 0.005, n = 10) in atherosclerosis+T2DM MSCs. (D) Representative example of flow cytometry proliferation. Histograms show CFSE dilution following in vitro proliferation of monocyte-depleted peripheral blood mononuclear cells (PBMCs) in co-culture with atherosclerosis+T2DM MSCs with or without the use of the IKKβ inhibitor MLN120B. (E) MLN120B improved the ability of atherosclerosis+T2DM MSCs to suppress the proliferation of activated T‐cells (***P = 0.0005, n = 12) while not affecting (F) CD4+ T cell viability (n = 12). (G and H) Similar immunopotency and CD4+ T cell viability in both cell–cell contact (n = 4) and trans-well conditions (n = 4) in MLN120B-treated atherosclerosis+T2DM MSCs. Each dot represents different biological samples (i.e. MSC from different donors). Mann–Whitney U test was used to compare two independent groups and Wilcoxon test was used for paired comparisons. Abbreviations: T2DM, type 2 diabetes mellitus; MSCs, mesenchymal stromal cells; IL 6, interleukin-6; IL-8, interleukin-8; MCP-1, monocyte chemoattractant protein-1; FSC‐A, forward scatter area; SSC‐A, side scatter area; SSC‐H, side scatter height; SSC‐W, side scatter width; 7-AAD, 7-aminoactinomycin D; EI, expansion index; CFSE, carboxyfluorescein succinimidyl ester.

Article Snippet: Six different human IKKβ guide RNAs (gRNAs) (see Supplementary material online , Table S1 ) and non-targeting gRNAs were purchased from GenScript (NJ, USA) and sub-cloned in pLentiCRISPR v2 using BsmBI restriction site (Addgene Plasmid No. 52961).

Techniques: Inhibition, In Vitro, Flow Cytometry, Co-Culture Assay, MANN-WHITNEY

Journal: Cardiovascular Research

Article Title: Ex vivo Ikkβ ablation rescues the immunopotency of mesenchymal stromal cells from diabetics with advanced atherosclerosis

doi: 10.1093/cvr/cvaa118

Figure Lengend Snippet: IKKβ knockdown in atherosclerosis+T2DM-MSCs enhances their immunoprotective effects in the permanent LAD ligation model. (A) Representative western blots showing the efficacy of two different gRNA molecules targeting the IKBKB locus in one control MSCs left either untreated or exposed to 10 ng/mL TNF-α for 10 min. Note that the KD efficiency was evaluated in all the MSCs samples used in vivo (see Supplementary material online, Figure S11). The effect of reducing the expression level of IKKβ in atherosclerosis+T2DM MSCs was evaluated on their (B) immunopotency in vitro (***P = 0.001, n = 11) and (C) ability to attract PBMCs (*P = 0.02, n = 4). (D) Haematoxylin and Eosin (H&E) staining of the injured (i.e. purple: infiltration of leucocytes) and viable myocardium (i.e. pink) (**P = 0.007, n = 8), (E) CD4+ (*P = 0.01, n = 7), (F) Monocyte/macrophage (MOMA-2 staining) (*P = 0.01, n = 7), and (G) FOXP3 (**P = 0.007, n = 8) content in heart sections and (H) IL-6 plasma levels (*P = 0.01, n = 7) were assessed. Each dot represents different animals injected with six different biological samples (i.e. MSC from different donors). Wilcoxon test used for paired groups Abbreviations: T2DM, type 2 diabetes mellitus; PBMCs, peripheric blood mononuclear cells; MOMA, monoclonal anti-macrophage antibody; IL 6, interleukin-6.

Article Snippet: Six different human IKKβ guide RNAs (gRNAs) (see Supplementary material online , Table S1 ) and non-targeting gRNAs were purchased from GenScript (NJ, USA) and sub-cloned in pLentiCRISPR v2 using BsmBI restriction site (Addgene Plasmid No. 52961).

Techniques: Knockdown, Ligation, Western Blot, Control, In Vivo, Expressing, In Vitro, Staining, Clinical Proteomics, Injection

Journal: EMBO Molecular Medicine

Article Title: Macrophage DCLK1 promotes atherosclerosis via binding to IKKβ and inducing inflammatory responses

doi: 10.15252/emmm.202217198

Figure Lengend Snippet: A Schematic illustration of quantitative proteomic screen to identify proteins binding to DCLK1. B 293T cells were transfected with Flag‐DCLK1 plasmid for 24 h. Control cells were transfected with empty vector (EV). Levels of Flag were measured by Western blot ( n = 3 biological replicates). C MS/MS spectrum of the peptide showing DLKPENIVLQQGEQR from DCLK1. D Co‐immunoprecipitation of DCLK1 and IKKβ in 293T cells transfected with Flag‐DCLK1. Flag‐DCLK1 was immunoprecipitated by anti‐Flag antibody. IgG, immunoglobulin G. E Co‐immunoprecipitation of DCLK1 and IKKβ in MPMs challenged with oxLDL (50 μg/ml) for 1 h. DCLK1 was immunoprecipitated by anti‐DCLK1 antibody. F MPMs isolated from DCLK1 f/f and DCLK1 MCKO mice were challenged with oxLDL (50 μg/ml) for 1 h. Western blot analysis and densitometric quantification of p‐IKKβ. GAPDH and IKKβ were used as loading controls ( n = 3 biological replicates). G 293T cells were transfected with IKKβ siRNA (si‐IKKβ) for 24 h, while control cells were transfected with negative control (NC) siRNA. Levels of IKKβ protein were measured by Western blot ( n = 3 biological replicates). H, I 293T cells were co‐transfected with Flag‐DCLK1 and si‐IKKβ for 24 h. Western blot analysis (H) and densitometric quantification (I) of IκBα, p‐IKKβ and p‐p65. GAPDH, IKKβ and p65 were used as loading controls ( n = 3 biological replicates). J 293T cells were co‐transfected with Flag‐DCLK1 and si‐IKKβ for 48 h. Protein levels of TNF‐α and IL‐6 were analyzed using ELISA ( n = 3 biological replicates). Data information: Data were shown as mean ± SEM; * P < 0.05; ns, not significant, two‐tailed unpaired Student's t ‐test. Source data are available online for this figure.

Article Snippet: The custom siRNA synthesized for human IKKβ (5′‐ GGAUUACAUUAGUGGACAATT‐3′) was purchased from RiboBio (Guangzhou, China).

Techniques: Binding Assay, Transfection, Plasmid Preparation, Control, Western Blot, Tandem Mass Spectroscopy, Immunoprecipitation, Isolation, Negative Control, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: Molecular Therapy. Nucleic Acids

Article Title: Inhibition of IKKβ/NF-κB signaling facilitates tendinopathy healing by rejuvenating inflamm-aging induced tendon-derived stem/progenitor cell senescence

doi: 10.1016/j.omtn.2021.12.026

Figure Lengend Snippet: NF-κB signaling is activated during RCT development (A) Western blotting for NF-κBp-p65 and NF-κBp65 and (B) quantification conforming the significantly increased NF-κB activation in S-TSPCs than that in H-TSPCs (n = 3 per group). (C) Immunofluorescence (green) staining and (D) quantification for NF-κBp65 revealing significantly increased nuclear NF-κBp65 localization in S-TSPCs than that in H-TSPCs. The red triangle indicates nuclear localization of NF-κBp65 (n = 3 per group). (E) Western blotting for NF-κBp-p65 and NF-κBp65 and (F) quantification conforming that IKKβ siRNA can significantly decrease NF-κB activation in IL-1β-induced senescent TSPCs (n = 3 per group). (G) Immunofluorescence (green) and (H) quantification for NF-κBp65 revealing that IKKβ siRNA can significantly decrease IL-1β-induced nuclear NF-κBp65 localization. The red triangle indicates nuclear localization of NF-κBp65 (n = 3 per group). (I) SA-β-gal staining and (J) quantification of senescent human TSPCs (n = 3 per group). (K) Immunofluorescence staining and (L) quantification of p16 INK4A (red) and p21 CIP1 (green) (n = 3 per group). NF-κBp-p65, phosphorylated NF-κB p65. NF-κBp65, unphosphorylated NF-κB p65. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Article Snippet: To determine the most effective IKKβ siRNA sequence, four different siRNA duplexes against human IKKβ and a nonsense siRNA (nsRNA) were purchased from GenePharma Biotechnology Company.

Techniques: Western Blot, Activation Assay, Immunofluorescence, Staining

Journal: Molecular Therapy. Nucleic Acids

Article Title: Inhibition of IKKβ/NF-κB signaling facilitates tendinopathy healing by rejuvenating inflamm-aging induced tendon-derived stem/progenitor cell senescence

doi: 10.1016/j.omtn.2021.12.026

Figure Lengend Snippet: Blocking IKKβ mitigates TSPC senescence (A) Heatmap representing the differential expression of 108 genes in IL-1β- and IL-1β + IKKβ siRNA-treated TSPCs. (B) Representative 10 downregulated pathways in IL-1β + IKKβ siRNA-treated TSPCs analyzed using the KEGG pathway. (C) Gene set enrichment analysis plots demonstrate that IL-1β- and IL-1β + IKKβ siRNA-treated TSPCs positively correlate with gene sets for stemness (BOQUEST STEM CELL UP), decreased cellular senescence (NEGATIVE REGULATION OF CELLULAR AGING), proliferation (STEM CELL PROLIFERATION), and decreased adipogenic potential (TSENG ADIPOGENIC POTENTIAL DN).

Article Snippet: To determine the most effective IKKβ siRNA sequence, four different siRNA duplexes against human IKKβ and a nonsense siRNA (nsRNA) were purchased from GenePharma Biotechnology Company.

Techniques: Blocking Assay, Expressing

Journal: Molecular Therapy. Nucleic Acids

Article Title: Inhibition of IKKβ/NF-κB signaling facilitates tendinopathy healing by rejuvenating inflamm-aging induced tendon-derived stem/progenitor cell senescence

doi: 10.1016/j.omtn.2021.12.026

Figure Lengend Snippet: Preparation and characterization of AuNCs and AuNC-siRNA (A) TEM images of AuNCs. (B) The mean diameter of AuNCs (n = 10 per group). (C) TEM images of AuNC-siRNA. (D) Mean diameter of AuNC-siRNA (n = 10 per group). (E) Surface charge of IKKβ siRNA, AuNCs, and AuNC-siRNA (n = 3 per group). (F) XPS for the C, N, O, S, P, and Au on AuNC and AuNC-siRNA surface. (G) Live/dead cell staining for TSPCs treated with different concentrations of AuNCs. Green fluorescence indicates live cells and red fluorescence indicates dead cells. (H) Quantification of TSPCs viability (n = 3 per group). (I) A Cell Counting Kit-8 assay for the effects of different AuNC concentrations on TSPCs proliferation (n = 3 per group). (J) The effects of different AuNCs concentrations on cell cycle distribution (n = 3 per group).

Article Snippet: To determine the most effective IKKβ siRNA sequence, four different siRNA duplexes against human IKKβ and a nonsense siRNA (nsRNA) were purchased from GenePharma Biotechnology Company.

Techniques: Staining, Fluorescence, Cell Counting

Journal: Molecular Therapy. Nucleic Acids

Article Title: Inhibition of IKKβ/NF-κB signaling facilitates tendinopathy healing by rejuvenating inflamm-aging induced tendon-derived stem/progenitor cell senescence

doi: 10.1016/j.omtn.2021.12.026

Figure Lengend Snippet: SiRNA protection and delivery efficiency of AuNC-siRNA (A) Free siRNA degradation by RNase. (B) SiRNA protection in AuNC-siRNA against RNase. (C) Free siRNA and AuNC-siRNA uptake in TSPCs. SiRNA was labeled with Cy5 dye (Cy5–siRNA). Scale bars, 20 μm. (D) qRT-PCR assay of IKKβ mRNA expression (n = 3 per group). (E) Western blotting assay of the levels of IKKβ protein. (F) Relative IKKβ protein quantification in TSPCs normalized to GAPDH expression (n = 3 per group). ∗∗p < 0.01.

Article Snippet: To determine the most effective IKKβ siRNA sequence, four different siRNA duplexes against human IKKβ and a nonsense siRNA (nsRNA) were purchased from GenePharma Biotechnology Company.

Techniques: Labeling, Quantitative RT-PCR, Expressing, Western Blot

Journal: Oncotarget

Article Title: Fas-associated factor 1 inhibits tumor growth by suppressing Helicobacter pylori-induced activation of NF-κB signaling in human gastric carcinoma

doi: 10.18632/oncotarget.14033

Figure Lengend Snippet: A . Quantitative RT-PCR analysis of FAF1 mRNA levels in HGC-27/FAF1 cells not transfected or transfected with negative-control RNAi (RNAi-NC) or with RNAi targeting IKKβor p65. Cells were infected or not with H. pylori . *P < 0.05 vs untransfected cells or cells transfected with RNAi-NC. B . Western blotting of total lysates from HGC-27/FAF1 cells not transfected or transfected with negative-control RNAi (RNAi-NC) or with RNAi targeting IKKβ or p65. Cells were infected or not with H. pylori . C . Western blotting of proteins in the NF-κB signaling pathway. D . Western blotting showed that levels of phosphorylated IKKα/β, p65 and IκBα were lower in RNAi targeting IKKβ or p65 than in untransfected cells or cells transfected with negative-control RNAi (P < 0.05). E . The ratio of IκBα to P-IκBα was higher in cells transfected with RNAi against IKKβ or p65 than in untransfected cells or cells transfected with negative-control RNAi (*P < 0.05). F . Secretion of TNF-α and IL-8 was lower in H. pylori -infected cells transfected with RNAi against IKKβ or p65 than in H. pylori -infected cells that were untransfected or transfected with negative-control RNAi (*P < 0.05). Data are the mean ± SD of three independent experiments.

Article Snippet: Constructs for RNA interference (RNAi) targeting the human NF-κB p65 subunit mRNA (GenBank, NM_021975) or human NF-κB IKKβ subunit mRNA (GenBank, NM_001190720) were also synthesized by Shanghai GeneChem.

Techniques: Quantitative RT-PCR, Transfection, Negative Control, Infection, Western Blot