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