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Figure 2. GDNF induces GDNF expression. A) Cells were treated without (Con) or with 50 ng/ml of recombinant GDNF polypeptide for the indicated period. GDNF expression was analyzed by RT-PCR. Histogram depicts the mean ratios of GDNF to Actin ± SD of 3 experiments. **P < 0.01, compared with control. B) SHSY5Y cells stably transfected with the pUSE empty vector were treated with conditioned media from the empty vector cells (CM-C) or media from the pUSE-GDNF stable cells (CM-GDNF) for the indicated length of time. Histogram depicts the mean ratios of GDNF to Actin ± SD of 3 experiments. *P < 0.05; **P < 0.01, compared with control (CM-C). C) pUSE cells were preincubated without (lanes 1 and 2) or with (lanes 3 and 4) 0.3 u/ml PI-PLC for 1 h. Cells were washed and incubated with media from pUSE cells (CM-C, lanes 1 and 3) or from pUSE-GDNF cells (CM-GDNF, lanes 2 and 4) for 3 h. Histogram depicts the mean ratios of GDNF to Actin ± SD of 3 experiments. **P < 0.01, lane 2 vs. 1, or lane 4 vs. 2. D) CM-C (lanes 1 and 3), and CM-GDNF (lanes 2 and 4) were preincubated for 1 h without (lanes 1 and 2), or with (lanes 3 and 4) with 10 µg/ml anti-GDNF neutralizing antibodies prior to incubation of pUSE cells with the indicated media for an additional 3 h. Histogram depicts the mean ratios of GDNF to Actin ± SD of 3 experiments. **P < 0.01, lane 2 vs. 1, or lane 4 vs. 2. E) pUSE cells were treated without (lanes 1, 3, 5, 7, 9, 11) or with (lanes 2, 4, 6, 8, 10, 12) CM-GDNF for 3 h. Cells were then washed and incubated in fresh media as indicated. Histogram depicts the mean ratios of GDNF to Actin ± SD of 3 experiments. *P < 0.05; **P < 0.01, compared with control (CM-C). F) pUSE cells were treated with CM-C or with CM-GDNF for 3 h. Cells were then extensively washed and incubated in fresh media for the indicated period without (lower panel) or with (upper panel) 5 µg/ml actinomycin D (A/D). Histogram depicts the mean percentages of GDNF ± SD of 3 experiments. **P < 0.01 compared with 0 time.
