Fig. 4: OGD/R-ADEXs regulate autophagy and provide neuroprotection using Nampt.
a The expression of Nampt in exosomes obtained from astrocytes was determined by western blotting. IDE was, internal marker for exosomes, used as negative markers (n = 5). b The expression of Nampt in OGD/R-ADEXs that were pretreated with either Protease K (+Protease), the Triton X-100 detergent (+Triton X), both of them (+Protease+Triton X), or with the solvent alone (Control) was determined by western blotting, which was normalized to IDE (n = 5). c The expressions of Nampt in neurons treated with OGD/R astrocytes or OGD/R-ADEXs were determined by western blotting (n = 5). d The cell viability was detected in OGD/R-induced neurons treated with PBS, OGD/R-ADEXs, OGD/R-ADEXssh-Nampt, or OGD/R-ADEXssh-NC using the CCK-8 assay (n = 5). e The expressions of LC3 II/I were determined by western blotting of OGD/R-induced neurons treated with PBS, OGD/R-ADEXs, OGD/R-ADEXssh-Nampt, or OGD/R-ADEXssh-NC. f, g Representative photomicrographs and quantitation of LC3-positive puncta in OGD/R-induced neurons treated with PBS, OGD/R-ADEXs, OGD/R-ADEXssh-Nampt, or OGD/R-ADEXssh-NC (n = 5). Scale bar: 10 μm. h, i The autophagosomes were detected by transmission electron microscopy of OGD/R-induced neurons treated with PBS, OGD/R-ADEXs, OGD/R-ADEXssh-Nampt, or OGD/R-ADEXssh-NC (n = 5). Scale bar: 1 μm. Data are shown as the mean ± SD of at least three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Neurons incubated under standard cell culture conditions (Control) were used as negative control; Neurons treated with PBS under OGD/R conditions served as positive control (OGD/R); ADEXs, exosomes isolated from astrocytes; OGD/R-ADEXs, exosomes isolated from OGD/R astrocytes; Co-culture, OGD/R neurons co-cultured with OGD/R astrocytes; OGD/R-ADEXssh-Nampt, exosomes isolated from OGD/R astrocytes that were pretreated with sh-Nampt; OGD/R-ADEXssh-NC, exosomes isolated from OGD/R astrocytes that were pretreated with scramble.
