Fig 2: Neuroprotective effect of hMSCs is attributed to the release of IGFBP-6 in LPC-treated organotypic spinal cord slice cultures. (A) hMSCs or hMSCs incubated with anti-IGFBP-6-Ab were transplanted into LPC-treated spinal cord slice cultures. Scale bar, 100 µm (B) Cell death was examined 7 days following LPC treatment by TUNEL staining. Arrows indicate fluorescence staining with TUNEL. The number of TUNEL-positive cells was quantified as the mean ± standard error of the mean of three independent experiments. **P<0.01 vs. control; ††P<0.01 vs. LPC treatment; ##P<0.01 vs. transplantation of hMSCs. Analysis of variance followed by the Newman-Keuls post hoc test were used. hMSC-CM, human mesenchymal stem cell-conditioned medium; LPC, lysolecithin; IGFBP-6 Ab, insulin-like growth factor binding protein 6 antibody; TUNEL, terminal deoxynuceotidyl transferase dUTP nick-end labeling; TP, transplantation.

Fig 3: IGFBP-6 activates Akt in oxidative stress-damaged primary cortical neurons via IGF-1 receptor-dependent signaling. (A) Phosphorylation of Akt was examined in primary cortical neurons incubated with hMSC-CM or hMSC-CM pretreated with an anti-IGFPB-6-Ab following H2O2 treatment. (B) Primary cortical neurons were pre-incubated with PPP (500 nM) for 15 min prior to H2O2 treatment. Expression levels of pAkt were detected 30 min following H2O2 treatment. Data are presented as the mean ± standard error of the mean of at least four independent experiments. **P<0.01, vs. control; ††P<0.01 vs. H2O2 (-) hMSC-CM; #P<0.05 and ##P<0.01 vs. H2O2 (+) hMSC-CM. Analysis of variance followed by the Newman-Keuls post hoc test were used. hMSC-CM, human mesenchymal stem cell-conditioned medium; pAKT, phosphorylated Akt; IGFBP-6 Ab, insulin-like growth factor binding protein 6 antibody; PPP, picropodophyllin.

Fig 4: IGFBP-6 inhibits extracellular IGF-2 induced by oxidative stress in primary cortical neurons. Concentrations of (A) IGF-1 and (B) IGF-2 in the supernatant of primary cortical neuron cultures incubated with hMSC-CM or hMSC-CM pretreated with anti-IGFPB-6-Ab following H2O2 treatment were analyzed using an enzyme-linked immunosorbent assay. The data are presented the mean ± standard error of the mean of at least four independent experiments. *P<0.05 and **P<0.01 vs. control; †P<0.05 vs. H2O2; #P<0.05 vs. hMSC-CM. Analysis of variance followed by the Newman-Keuls post hoc test were used. hMSC-CM, human mesenchymal stem cell-conditioned medium; IGF, insulin-like growth factor; IGFBP-6 Ab, insulin-like growth factor binding protein-6 antibody.

Fig 5: IGFBP-6 modulates the translocation of Bax in primary cortical neurons. (A) Immunoblotting and quantification of cytosolic and mitochondrial protein levels of Bax in primary cortical neurons incubated with hMSC-CM or hMSC-CM pretreated with anti-IGFPB-6 antibody following H2O2 treatment. (B) Mitochondrial and (C) cytosolic levels of Bax were quantified 24 h following H2O2 treatment. The data are presented the mean ± standard error of the mean of at least four independent experiments. **P<0.01 vs. control; ††P<0.01 vs. H2O2; ##P<0.01 vs. hMSC-CM. Analysis of variance followed by the Newman-Keuls post hoc test were used. hMSC-CM, human mesenchymal stem cell-conditioned medium; cyto, cytosolic; mito, mitochondrial; Bax, B-cell lymphoma 2-like protein 4; COX-IV, cyclooxygenase IV; IGFBP-6 Ab, insulin-like growth factor binding protein-6 antibody.