Journal: Molecular and cellular neurosciences

Article Title: Direct transcriptional induction of Gadd45gamma by Ascl1 during neuronal differentiation.

doi: 10.1016/j.mcn.2010.03.014

Figure Lengend Snippet: Fig. 2. Optimization of growth conditions for differentiation. (A–H) Map2 (red) staining of P19T1A2 cells treated±Dox at 0.5 μg/ml for 10 days under the following growth conditions: (1) MEMα (7.5% CS, 2.5% FBS), (2) MEMα (1% FBS), (3) MEMα (7.5% CS, 2.5% FBS) for the first three days of differentiation, followed by a switch to Neurobasal media (B27, GlutaMAX), and (4) OPTI-MEM (1% FBS). Nuclei were visualized with DAPI and appear blue. Under the first three growth conditions, cells differentiated into neurons that were immunoreactive to Map2 in the presence of Dox (E–G), although cells grown in reduced serum were immunoreactive to Map2 protein in the absence of Dox (B). Cells grown in OPTI- MEM resulted in Map2-positive cells that were morphologically less differentiated than the other conditions (H). Scale bar=100 μm. (I) Western blotting for expression of Map2 protein under the growth conditions shown in panels A–H. The highest amount of Map2 protein was observed in cells cultivated in Neurobasal media (Condition 3).

Article Snippet: The following primary antibodies were used in the experiments: TetR, Tau (Chemicon), GAPDH, Map2 (Cell Signaling Technology), Ascl1 (BD Pharmingen), TuJ1 (Covance), Gap43 (Sigma-Aldrich), Isl1 (DSHB University of Iowa), Synaptophysin (Syp; BD Biosciences), and Gadd45γ (Sigma-Aldrich).

Techniques: Staining, Western Blot, Expressing

Journal: Molecular and cellular neurosciences

Article Title: Direct transcriptional induction of Gadd45gamma by Ascl1 during neuronal differentiation.

doi: 10.1016/j.mcn.2010.03.014

Figure Lengend Snippet: Fig. 3. Time course of differentiation with P19T1A2 cells. (A) Map2 (green) staining of P19T1A2 cells treated±Dox at 0.5 μg/ml for the indicated days. Nuclei were visualized with DAPI and appear blue. In the absence of Dox, no Map2-positive cells were observed. In the presence of Dox, Map2-positive cells became evident after three days of treatment with Dox. Cells expressing Map2 underwent neuritogenesis by day five, and by day eight, the somas of Map2-positive cells began to cluster together, while the neurites became elongated and better defined. Scale bar=100 μm. (B) Western blot for expression of Map2 and Ascl1 protein during the time course of differentiation of P19T1A2 cells. (C) Western blot for expression of cleaved PARP protein in P19T1A2 cells.

Article Snippet: The following primary antibodies were used in the experiments: TetR, Tau (Chemicon), GAPDH, Map2 (Cell Signaling Technology), Ascl1 (BD Pharmingen), TuJ1 (Covance), Gap43 (Sigma-Aldrich), Isl1 (DSHB University of Iowa), Synaptophysin (Syp; BD Biosciences), and Gadd45γ (Sigma-Aldrich).

Techniques: Staining, Expressing, Western Blot

Journal: Molecular and cellular neurosciences

Article Title: Direct transcriptional induction of Gadd45gamma by Ascl1 during neuronal differentiation.

doi: 10.1016/j.mcn.2010.03.014

Figure Lengend Snippet: Fig. 5. P19T1A2 cells show characteristics of mature neurons. (A) Western blot analysis examining the protein expression of selected Ascl1-induced target genes identified in the microarrays. As predicted from the microarray results, expression of Gap43, Isl1, Synaptophysin (Syp), and Tau proteins increased in response to Dox-induced overexpression of Ascl1. (B) P19T1A2 cells have the electrophysiological properties of neurons. Shown is a representative action potential-like waveform recorded from a P19T1A2 cell grown in the presence of Dox for six days. (C, C’) P19T1A2 cells are polarized. Immunostaining for expression of the dendritic marker Map2 (red) and the axonal marker Neurofilament-L (NF-L, green). Nuclei were visualized with DAPI staining and appear blue. The boxed area in panel C is enlarged in panel C’, with dendritic varicosities indicated by white arrowheads.

Article Snippet: The following primary antibodies were used in the experiments: TetR, Tau (Chemicon), GAPDH, Map2 (Cell Signaling Technology), Ascl1 (BD Pharmingen), TuJ1 (Covance), Gap43 (Sigma-Aldrich), Isl1 (DSHB University of Iowa), Synaptophysin (Syp; BD Biosciences), and Gadd45γ (Sigma-Aldrich).

Techniques: Western Blot, Expressing, Microarray, Over Expression, Immunostaining, Marker, Staining