Fig. 3: MSC-secreted exosomes inactivate NF-κB pathway. | Cell Death & Disease

Fig. 3: MSC-secreted exosomes inactivate NF-κB pathway.

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

Fig. 3

a Luciferase reporter assay detected the luciferase activity of Ikbkb promoter vector under coculture of MSC. b, c The relative mRNA level of Dicer and Ikbkb was examined by RT-qPCR in LPS-treated MLE-12 cells cocultured with MSC or Dicer-silenced MSC, respectively. d The protein levels of NF-κB pathway key factors (IKKβ, p-IκBα, and p-IKBβ in the whole cell lysate as well as the nuclear protein level of p65) were checked using western blot after cocultured with MSC or Dicer-silenced MSC. e Electron microscope analyzed the existence of exosomes secreted by MSC or Dicer-silenced MSC. f PKH67 staining was used to confirm the entrance of exosomes secreted by MSC or Dicer-silenced MSC into target cells. Scale bar = 100 μm. g The diameter of exosomes was measured and identified through NTA. h The surface markers of exosomes (CD9, CD63, CD81, and HSP70) were detected by western blot analysis. i RT-qPCR examined the level of Ikbkb mRNA in LPS-treated MLE-12 cells treated with MSC-exosome or MSC/sh-Dicer-exosome. j Western blot examined the protein levels of nuclear p65, Ikkβ, p-IκBα, and p-IκBβ in LPS-treated MLE-12 cells treated with MSC-exosome or MSC/sh-Dicer-exosome. k IF staining detected the nuclear translocation of p65 in LPS-treated MLE-12 cells under the context of MSC-exosome or MSC/sh-Dicer-exosome. Scale bar = 50 μm. l The cellular location of p65 was validated by western blot analysis in LPS-treated MLE-12 cells with MSC-exosome or MSC/sh-Dicer-exosome treatment. **p < 0.01. n.s. no statistical significance.

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