Fig 1: Identification of IKBKB as a direct target of miR-214–3p in chondrocytes. (a) Venn diagram display the overlapping of the human target genes of miR-214–3p, as predicted by Targetscan, miRDB, starbase and NF-κB core genes. (b) Immunofluorescence analysis (left) and quantification data (right) of IKKβ of human cartilage in undamaged and damaged areas in OA patients. (c, d) qRT-PCR analysis of IKBKB in human chondrocytes transfected with miR-214–3p inhibitor (c) or miR-214–3p mimics (d) with or without IL-1β stimulation. (e, f) Western blotting analysis of IKKβ protein levels in chondrocytes after miR-214–3p inhibition (e) or overexpression (f). The data were normalized by GAPDH. (g) Representative images of IKKβ assayed by immunofluorescence confocal microscopy in human chondrocytes with miR-214–3p knockdown or overexpression with IL-1β. DAPI, 4′,6-diamidino-2-phenylindole. Scale bar, 25 μm. (h) Sequence alignment of a putative miR-214–3p binding site within the 3′-UTR of IKBKB mRNA shows a high level of sequence conservation and complementarity with miR-214–3p. (i) HEK-293T cells were co-transfected with miR-214–3p mimics or scramble and luciferase reporter constructs of the wild-type IKBKB-3′UTR (3′UTR-wt) or the mutated IKBKB-3′UTR (3′UTR mut). Luciferase activity was measured after transfection. Luciferase reporter assay revealed that miR-214–3p exclusively decreased luciferase activity of the wild-type reporter plasmids. ns: no significant difference, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. All data are shown as means ± SEM of three independent experiments in (b), (c), (d) and (i). Student's t-test and one-way ANOVA were used for comparison between two groups and multiple groups, respectively.
Fig 2: MSC-secreted exosomes inactivate NF-κB pathway.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.
Fig 3: MiR-182-5p transmitted by MSC-exosomes reverses EMT process by directly targeting Ikbkb.a Online starBase v2.0 predicted 71 miRNAs targeted to Ikbkb were subjected to RT-qPCR analysis in LPS-treated MLE-12 cells under MSC coculture or not. b RT-qPCR analyzed the level of miR-182-5p in LPS-treated MLE-12 cells treated with MSC-exosome. c Relative level of miR-182-5p was detected by RT-qPCR in LPS-treated MLE-12 cells transfected with specific miRNA mimics. d Flow cytometry analysis displayed the apoptosis rate in LPS-treated MLE-12 cells transfected with miR-182-5p mimics. e IF assay analyzed the fluorescence intensities of two EMT-related proteins, E-cadherin, and Vimentin, in LPS-treated MLE-12 cells transfected with miR-182-5p mimics. Scale bar = 50 μm. f, g RT-qPCR and western blot examined the levels of epithelial marker (E-cadherin) and mesenchymal markers (α-SMA, TGF-β1, Collagen type I, and Collagen type III) in LPS-treated MLE-12 cells transfected with miR-182-5p mimics. h The binding sites between miR-182-5p and Ikbkb were predicted. i Luciferase reporter assay examined the luciferase activity of indicated reporter vectors in LPS-treated MLE-12 cells and HEK-293T cells co-transfected with miR-182-5p mimics or NC mimics. j IF assay detected p65 nuclear translocation in LPS-treated MLE-12 cells with MSC-exosome or MSC-exosome+miR-182-5p inhibitor. Scale bar = 50 μm. k Western blot analysis detected cytoplasmic and nuclear p65 in LPS-treated MLE-12 cells with MSC-exosome or MSC-exosome+miR-182-5p inhibitor. l RT-qPCR and western blot examined the mRNA and protein levels of Ikbkb in LPS-treated MLE-12 cells transfected with MSC-exosome or MSC-exosome+miR-182-5p inhibitor. **p < 0.01. n.s. no statistical significance.
Fig 4: NF-κB and hedgehog signaling pathways are inactivated in LPS-treated MLE-12 cells cocultured with MSC.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.
Fig 5: MSC-exosome delivered miR-182-5p and miR-23a-3p to mitigate LPS-induced ALI and pulmonary fibrosis by targeting Ikbkb/Usp5 signaling.a HE staining of lung tissues in four different groups (LPS, LPS + MSC-exosome, LPS + MSC-exosome+miR-23a-3p inhibitor, LPS + MSC-exosome+miR-23a-3p inhibitor+miR-182-5p inhibitor). Scale bar = 200 µm. b The concept map demonstrating the role and functional mechanism of MSC on alleviating ALI and pulmonary fibrosis. *p < 0.05, **p < 0.01.
Supplier Page from Abcam for Anti-IKK beta antibody [EPR6043]