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. 2011 Sep 7;31(36):12802–12815. doi: 10.1523/JNEUROSCI.0898-11.2011

Figure 3.

Figure 3.

GDNF-mediated induction of Pitx3 expression activates BDNF transcription. a, b, RT-PCR (a) and Western blot (b) analyses of untreated (first lane), GDNF-treated (second lane), and SN50 + GDNF-treated (third lane) E14 rat primary VM cultures showed increased Pitx3 and BDNF expression after GDNF-treatment, which was blocked by simultaneous treatment with NF-κB inhibitor SN50. c, qPCR analysis indicated an 1.3-fold increase of Pitx3 and 1.4-fold increase of BDNF mRNA levels after GDNF-treatment and 70% reduction in both cases after SN50 application (Pitx3: GDNF, 2.27 ± 0.36; GDNF + SN50, 1.40 ± 0.09, mean ± SEM.; BDNF: GDNF, 2.35 ± 0.35; GDNF + SN50, 1.40 ± 0.11, mean ± SEM; *p < 0.05; **p < 0.005 in the one-way ANOVA for repeated measurements; n = 4). Con, Control. d, e, RT-PCR (d) and Western blot (e) analyses of untreated (first lane), GDNF-treated (second lane), Pitx3 siRNA + GDNF-treated (third lane), and control siRNA + GDNF-treated (fourth lane) E14 rat primary VM cultures revealed that siRNA-mediated knockdown of Pitx3 (siRNA), but not a control siRNA (CRNA) abolished the increased expression of BDNF in these cultures after GDNF-treatment. f, qPCR analysis indicated that Pitx3 siRNA + GDNF treatment resulted in a 56 and 32% reduction of Pitx3 and BDNF mRNA levels, respectively, as compared to GDNF only-treated cultures and a control siRNA (Pitx3: GDNF, 2.53 ± 0.33; GDNF + Pitx3 siRNA, 1.12 ± 0.02; GDNF + control siRNA, 2.21 ± 0.22, mean ± SEM; BDNF: GDNF, 1.73 ± 0.12; GDNF + Pitx3 siRNA, 1.18 ± 0.09; GDNF + control siRNA, 1.59 ± 0.11, mean ± SEM; **p < 0.005 in the one-way ANOVA for repeated measurements). Data were derived from at least three independent experiments in each case.