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: Antioxidants

Article Title: Gastro-Protective Effects of Albizia anthelmintica Leaf Extract on Indomethacin-Induced Gastric Ulcer in Wistar Rats: In Silico and In Vivo Studies

doi: 10.3390/antiox10020176

Figure Lengend Snippet: Effects of indomethacin alone and with oral pre-treatments of famotidine or A. anthelmintica (200, 100 mg/kg) on ( A ) gastric inhibitor of nuclear factor kappa-B kinase subunit beta (IKKB), ( B ) gastric nuclear factor KB (NF-κB), ( C ) gastric TNF-α, and ( D ) gastric IL-6 in rats. Statistical analyses were carried out by one-way ANOVA followed by Tukey’s post hoc test, mean ± SEM. * Significantly different from control group at p < 0.05. @ Significantly different from indomethacin alone group at p < 0.05.

Article Snippet: Primary rabbit polyclonal antibodies against inhibitor of nuclear factor kappa-B kinase subunit beta (IKK-β, catalogue number: PA2036-1) and rabbit monoclonal antibodies against β-actin (catalogue number: M01263) were purchased from Boster Biological Technology (Pleasanton, CA, USA) and diluted at 1:1000 and 1:5000, respectively.

Techniques: Control

Journal: Cells

Article Title: Salidroside Derivative SHPL-49 Exerts Anti-Neuroinflammatory Effects by Modulating Excessive Autophagy in Microglia

doi: 10.3390/cells14060425

Figure Lengend Snippet: SHPL-49 exerts anti-inflammatory effects by suppressing the NF-κB signaling pathway. ( A ) Immunohistochemical staining images of NF-κB and IL-6 in brain tissue sections from rats subjected to pMCAO and treated with SHPL-49 (15 mg/kg) and ED (7.5 mg/kg) for three days, Scale = 100 μm. ( B ) The IODs for NF-κB and IL-6 immunohistochemistry in brain tissue sections for each treatment group. ( C ) Representative Western blot of NF-κB in the nucleus and cytoplasm of OGD-BV2 cells after treatment with SHPL-49 (200 μM). ( D ) Quantification of the Western blot results. ( E ) Immunofluorescence staining pattern of NF-κB in OGD-BV2 cells following treatment with SHPL-49 (200 μM); Scale = 10 μm. ( F ) Quantification of the average fluorescence intensity of NF-κB in the nucleus of BV2 cells. ( G ) Representative Western blot of p-IKKβ, IKKβ, p-IκBα, and IκBα proteins in OGD-BV2 cells after treatment with SHPL-49 (200 μM). ( H ) Quantification of the Western blot results. ( I ) The protein expression levels of inflammatory factors IL-6, IL-1β, and iNOS in the supernatant of OGD-BV2 cells following treatment with SHPL-49 (200 μM). Data are presented as means ± SD with n = 6 per group. *** p < 0.001 vs. Ctrl; ## p < 0.01, ### p < 0.001 vs. OGD.

Article Snippet: Membranes were incubated with 5% nonfat milk (Beyotime, Shanghai, China, P0216) or BSA (Beyotime, Shanghai, China, ST023) followed by overnight incubation at 4 °C with the following primary antibodies: NF-κB (1:1000, Cell Signaling Technology, MA, USA, D14E12), p-IKKβ (1:1000, UpingBio, Hangzhou, Zhejiang, China, YP-Ab-14443), IKKβ (1:1000, Boster, Wuhan, China, BM4875), p-IκBα (1:1000, UpingBio, Zhejiang, China, YP-Ab-01253), IKBα (1:1000, UpingBio, Zhejiang, China, YP-Ab-01830), P62/SQSTM1 (1:1000, Boster, Wuhan, China, BM4385), LC3B (1:200, PTMab, Zhejiang, China, PTM-6384), ATG5 (1:1000, Cell Signaling Technology, MA, USA, D5F5U), Bclin1 (1:1000, Cell Signaling Technology, MA, USA, D40C5), LAMP2 (1:200, UpingBio, Zhejiang, China, YP-Ab-14094), Bax (1:1000, Cell Signaling Technology, MA, USA, 14796), Bcl-2 (1:1000, AiFang biologic, Hunan, China, AF0060), Cleaved caspase-3 (1:1000, Cell Signaling Technology, MA, USA, 9661S), and Cleaved caspase-9 (1:1000, Cell Signaling Technology, MA, USA, 9507S).

Techniques: Immunohistochemical staining, Staining, Immunohistochemistry, Western Blot, Immunofluorescence, Fluorescence, Expressing

Journal: Scientific reports

Article Title: The N-Acetyl Phenylalanine Glucosamine Derivative Attenuates the Inflammatory/Catabolic Environment in a Chondrocyte-Synoviocyte Co-Culture System.

doi: 10.1038/s41598-019-49188-9

Figure Lengend Snippet: Figure 5. IL-1β increases IKKα nuclear translocation and phosphorylation of serine 10 on histone H3, while NAPA attenuates these signalling events. (A) Overlapping signals of nuclear counterstaining (DAPI) and IKKα detected via an Alexa Fluor 555 secondary antibody: the colocalized signals indicate that NAPA addition is effective in reducing nuclear translocation of IKKα. (B) Upper pictures: left, western blot of anti- phosphorylated serine 10 of histone H3, along with GAPDH as a loading control and right: densitometric analysis of the signal showing the different pattern of H3pSer10 accumulation in CTR (circle), IL-1β (square) or IL-1β + NAPA (triangle) conditions. Lower images: specificity of the signal obtained with the anti-H3 phosphorylated serine 10: 20x field pictures of chondrocytes grown on coverslips in the bottom of wells at time 0 in control (upper row) or IL-1β stimulated conditions (lower row): Green: IKKα detected with an Alexa Fluor 488 anti-rabbit antibody; red: H3pSer10 signal detected with an Alexa Fluor 555 anti-mouse antibody; blue: nuclear DNA stained with Hoechst 33342 and merged images.

Article Snippet: IKKα staining was performed with 5 μg/ml rabbit anti-IKKα antibody (BOSTER Biological Technology, Freemont, CA, USA), while the extent of phosphorylation of serine 10 of histone H3 was evaluated with 5 μg/ml anti-phospho-histone H3, (Ser10), (mouse monoclonal, Upstate–Millipore) overnight at 4 °C.

Techniques: Translocation Assay, Phospho-proteomics, Western Blot, Control, Staining