p-CREB is involved in ES-induced BDNF mRNA transcription and neurite outgrowth.

(A) CREB expression in cells transduced with control or CREB shRNA (mean ± SD; n = 3). β-tubulin was used as a loading control. (B, C) p-CREB expression in transduced cells with or without ES (mean ± SD; n = 3). Histone was used as a loading control. *P < 0.001 compared to ES-/Control shRNA; ^#P < 0.001 compared to ES+/Control shRNA; F-value = 49.293. (D) Relative BDNF mRNA levels in cells of the four groups (B) (mean ± SD; n = 4). β-actin was used as a loading control. *P < 0.001 compared to ES-/Control shRNA; ^#P < 0.05 compared to ES+/Control shRNA; ^&P < 0.05 compared to ES-/CREB shRNA; F-value = 37.889. (E) Typical images of neurite outgrowth in transduced cells with or without ES. β-tubulin III was used to indicate cell bodies and neuritis. Scale bar (100 μm) applies to a—d. (F) Quantification of neurite outgrowth in treated cells (E) (mean ± SD; n = 3). The assay was performed in triplicate. Numbers of cell neurites analyzed in each group: (a) 114, (b) 108, (c) 117, (d) 96. *P < 0.05 to ES-/Control shRNA; ^#P < 0.05 compared to ES+/Control shRNA; ^&P < 0.05 compared to ES-/CREB shRNA; F-value = 10.190. (G) Relative BDNF protein levels in cells of the four groups as (B) (mean ± SD; n = 4). *P < 0.001 compared to ES-/Control shRNA; ^#P < 0.05 compared to ES+/Control shRNA; ^&P < 0.05 compared to ES-/CREB shRNA; F-value = 20.396.