Fig 1: SRC kinases are activated by TNFa and BMP6, and can regulate Notch.(a) Schematic depicting SRC phosphorylation. (b) Representative immunoblots of phospho-SRC Tyr527, Tyr416 and total SRC expression in human dPASMCs from disease-free controls and HPAH patients treated with TNFa (1 ng ml-1) and/or BMP2 (10 ng ml-1) or BMP6 (10 ng ml-1) for 30 min as indicated. Reprobed for a-tubulin to ensure equal loading. The arrows indicate the SRC bands. The data shown are representative of three control and HPAH cell lines. (c) Representative immunoblots of phospho-SRC Tyr527, Tyr416 and total SRC expression in human control dPASMCs following transfection with DharmaFECT1 alone (DH1), siBMPR2, or non-targeting siRNA control (siCP) and treated with TNFa (1 ng ml-1) and/or BMP6 (10 ng ml-1) for 30 min as indicated. Reprobed for a-tubulin to ensure equal loading. The data shown are representative of three experiments. (d) Representative immunoblots of phospho-SRC Tyr527, Tyr416 and total SRC expression in human HPAH dPASMCs following transfection with DH1 alone, siACVR2A, or siCP and treatment with TNFa (1 ng ml-1) and/or BMP6 (10 ng ml-1) for 30 min as indicated. Reprobed for a-tubulin to ensure equal loading. The data shown are representative of three cell lines. (e,f) NOTCH2 and NOTCH3 mRNA expression in human HPAH dPASMCs following transfection with DH1 alone, siFYN, siYES, siSRC or siCP and treatment with TNFa (1 ng ml-1) or BMP6 (10 ng ml-1) for 1 h as indicated. Expression was normalized to ACTB (n=3). (g) Proliferation of human HPAH dPASMCs on day 6 following transfection with DH1 alone, siFYN, siYES, siSRC or siCP and treatment every 48 h with TNFa (1 ng ml-1) and/or BMP6 (50 ng ml-1) as indicated (n=3). (h) Proliferation of human control dPASMCs on day 6 following transfection with DH1 alone, siFYN, siYES, siSRC or siCP and treatment every 48 h with TNFa (1 ng ml-1) and/or BMP6 (50 ng ml-1) as indicated (n=3). One-way analysis of variance with post hoc Tukey's for multiple comparisons used in e,f,g and h. *P=0.05, **P=0.01, ***P=0.001. Error bars represent mean±s.e.m.
Fig 2: Exosomal miR-150-5p targets NOTCH2 in microglial cells. A StarBase predicted the miRNA-binding sites on NOTCH2; B RT-qPCR detected the expression of NOTCH2-binding miRNAs in BMSC-exosomes; C RT-qPCR detected the expression of the NOTCH2-binding miRNAs in microglial cells that were incubated with BMSC-exosomes or not; RNA pull down assay D and dual-luciferase reporter assay E verified the targeting relationship between miR-150-5p and NOTCH2; RT-qPCR F and western blotting G were used to detect the expression of NOTCH2 in microglial cells overexpressed or underexpressed miR-150-5p. Data were shown as mean ± standard deviation. Cellular experiments were repeated thrice. *P < 0.05, vs. the mimic NC or CM group; #P < 0.05, vs. the inhibitor NC group; &P < 0.05, vs the NC-probe group. BMSC bone marrow mesenchymal stem cell
Fig 3: BMSC-exosomes downregulate microglial NOTCH2 expression. RT-qPCR (A) and western blots (B) were used to detect NOTCH2 expression in microglial cells after BMSC-exosome treatment; RT-qPCR (C), western blotting (D), and immunohistochemistry (E) were used to detect the expression of NOTCH2 in SDH; (F) Double immunofluorescence staining was used to localize NOTCH2. Data were shown as mean ± standard deviation. N = 6. *P < 0.05, vs. the sham group; #P < 0.05, vs. the SNL group. BMSC bone marrow mesenchymal stem cell, SDH spinal dorsal horn, SNL spinal nerve ligation
Fig 4: Inhibition of miR-150-5p in BMSC-exosomes offsets the therapeutic effects of BMSC-exosomes on mechanical allodynia. A The transfection efficiency was observed under a fluorescence microscope; B RT-qPCR was used to detect the expression of miR-150-5p in BMSCs and BMSC-exosomes. RT-qPCR C and western blotting D were used to detect the expression of miR-150-5p and NOTCH2 in the SDH of SNL rats; Hind paw PWT E and PWL F of SNL rats were measured on days 0, 1, 3, 5, 10, 15, and 20 after surgery; G TUNEL staining revealed the apoptosis in the SDH; H ELISA detected the expression levels of TNF-a, IL-1ß and IL-6 in the SDH. Data were shown as mean ± standard deviation. N = 6. &P < 0.05, vs. the vector or Exo-vector group. BMSC bone marrow mesenchymal stem cell, SNL spinal nerve ligation, SDH spinal dorsal horn, PWT paw withdrawal threshold, PWL paw withdrawal latency
Fig 5: BMSC-exosomes suppress mechanical allodynia in rats by inhibiting the expression of NOTCH2. RT-qPCR (A) and western blotting (B) were used to detect the expression of NOTCH2 in rat SDH; Hind paw PWT (C) and PWL (D) of SNL rats were measured on days 0, 1, 3, 5, 10, 15, and 20 after surgery; (E) TUNEL staining revealed the apoptosis in SDH; (F) ELISA detected the expression levels of TNF-a, IL-1ß, and IL-6 in the SDH. Data were shown as mean ± standard deviation. N = 6. *P < 0.05, vs. the sh-NC group; #P < 0.05, vs. the pcDNA3.1 group; &P < 0.05, vs. the pcDNA3.1-NOTCH2 group. BMSC bone marrow mesenchymal stem cell, SNL spinal nerve ligation, SDH spinal dorsal horn, PWT paw withdrawal threshold, PWL paw withdrawal latency
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