Figure 3

Transcription and secretion of BMP2 and FGF2 were increased in hADMPCs exposed to oxidative stress. (A, B) Upregulation of BMP2 (A) and FGF2 (B) mRNA in hADMPCs by BSO in a dose-dependent manner. (C, D) Secretion of BMP2 (C) and FGF2 (D) from hADMPCs in medium alone (cont) or with addition of 1 mM BSO (BSO) was analyzed by ELISA. (E, F) NAC treatment repressed the expression levels of BMP2 and FGF2 upregulated by BSO to the control levels. Expression of BMP2 (E) and FGF2 (F) mRNA was analyzed by q-PCR. cDNA was generated from total RNA extracted from hADMPCs (cont), hADMPCs treated with 1 mM BSO (BSO), 1 mM BSO + 5 mM NAC (BSO + NAC), and 5 mM NAC (NAC). The most reliable internal control gene was determined using the geNorm Software. (G, H) PC12 cells were cultured in differentiation medium alone (control), or differentiation medium supplemented with BMP2 (40 ng/mL), FGF2 (5 ng/mL), or both BMP2 and FGF2 (BMP2 + FGF2) for 2 days. (G) Representative images of neurite outgrowth in PC12 cells. Scale bars, 200 μm. (H) One hundred individual neurites were measured in each sample using Dynamic Cell Count Analyzer BZ-H1C (Keyence) and average neurite length was calculated. *, P < 0.05 (Student’s t test). (I, J) PC12 cells were cultured in CM-BSO (−), CM-BSO (+), or CM-BSO (+) added with recombinant murine Noggin (200 ng/mL). (I) Western blot analysis of PC12 cells 1 h after CM treatment. Proteins extracted from each sample were resolved by SDS-PAGE, transferred to a membrane, and probed with anti-phosphorylated Smad1/5/8 (phospho-Smad1/5/8) and anti-Actin. Numbers below blots indicate relative band intensities as determined by the ImageJ software. (J) Two days after CM treatment, 100 individual neurites in PC12 cells were measured in each sample using Dynamic Cell Count Analyzer BZ-H1C (Keyence) and average neurite length was calculated. *, P < 0.05 (Student’s t test).