Contribution of ERK- and cAMP-dependent PKA signaling pathways to luteolin–induced miR-132 up-regulation and neurite outgrowth in PC12 cells.

<p>PC12 cells were seeded on poly-L-lysine-coated 6-well plates in normal medium for 24 h. Cells were then shifted to low-serum medium (1% horse serum and 0.5% FBS) and then pretreated with the inhibitors U0126 (10 µM), H-89 (10 µM) or SQ22536 (500 µM) for 30 min followed by exposure to vehicle or luteolin (20 µM) for 2 h. Cellular RNA was then prepared, and the levels of immature miR-132 (pri- and pre-miR-132) (A) and mature miR-132 (B) was detected by reverse transcription quantitative PCR as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043304#s4" target="_blank">Materials and Methods</a>. Data represent the mean ± SD from three independent experiments. **<i>p</i><0.01 represents significant differences compared to vehicle-treated cells. ##<i>p</i><0.01 represents significant differences compared to the respective inhibitor-non-treated group. (C) PC12 cells were seeded on poly-L-lysine-coated 6-well plates in normal medium for 24 h. Cells were then shifted to low-serum medium (1% horse serum and 0.5% FBS) for 24 h and then pre-treated for 30 min with the indicated inhibitors followed by exposure to vehicle or luteolin (20 µM) for 72 h. Neurite-bearing cells were analyzed as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043304#s4" target="_blank">Materials and Methods</a>. (D) The average maximal neurite length for each of the differentiated cells was analyzed by Image J software. Data represent the mean ± SD from three independent experiments. **<i>p</i><0.01 represents significant differences compared to vehicle-treated cells. ##<i>p</i><0.01 represents significant differences compared to the respective inhibitor-non-treated group. (E) PC12 cells were transfected with miR-132 mimics (miR-132) or miRNA mimics negative control (miR-NC) for 24 h as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043304#s4" target="_blank">Materials and Methods</a>. For the treatment of cells with the inhibitor, transfected cells were pre-incubated with indicated inhibitors for 30 min and then exposed to luteolin (20 µM) for an additional 72 h. Neurite-bearing cells were analyzed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043304#s4" target="_blank">Materials and Methods</a>. Data represent the mean ± SD from three independent experiments. *<i>p</i><0.05 and **<i>p</i><0.01 represent significant differences compared to miR-NC transfected group.