Figure 1.
Treatment scheme for chronic influence of growth factors on expression of GABA cell marker.
Cells were maintained for two days under proliferation conditions (undifferentiated (undiff) as shown in the left picture). For the first 4 days of differentiation, a routine differentiation protocol for HiB5 was followed (see methods section): after two days at 39°C in N2 medium, PDGF (30 ng/ml) was added and the cells incubated for another 2 days. To study the influence of growth factors they were added at day 4 and cells incubated for another 2 days.
Figure 2.
Screening of growth factors for GAD67 increasing activity in differentiated HiB5 cells.
Cultured HiB5 cells were switched to differentiation conditions. In the presence of PDGF, the cultured cells were treated with NRG-1 or VEGF and cells were incubated for 2 additional days. Controls were cultured in N2 medium with PDGF alone. Cells were harvested in Trizol, the total RNA extracted and subjected to quantitative RT-PCR. Data are expressed as fold change of N2 control. (*≤0.001 vs undiff).
Figure 3.
Expression pattern of mature neuron markers acetylated tubulin, MAP2 and PSD-95 and stem cell marker nestin in undifferentiated and differentiated HiB5 cells.
Specific markers were detected using primary antibodies and immunofluorescence detection with DAPI as nuclear counter stain. Detectable levels of IF were associated with, acetylated tubulin (A); MAP2 (B); PSD-95 in differentiated HiB5 cells (C), while nestin (D) is only seen in undifferentiated cells.
Table 1.
Quantification of cells expressing differentiation stage-specific markers.
Figure 4.
Induction of expression of key GABA cell markers GAD67 and GAT-1 in differentiated HiB5 cells.
(A, B): Cells were differentiated at 39°C in N2 for two days, followed by addition of PDGF (30 ng/ml) for an additional two days. This was followed by stimulation for two days with growth factors. Quantitative (q) RT-PCR for GAD67 or GAT1 was used to measure changes in mRNA expression in the presence N2 supplement plus PDGF alone; additional PDGF (P); or a combination of additional PDGF and BDNF (B; 50 and 100 ng/ml respectively). Values are expressed as fold increases in differentiated cells compared with undifferentiated controls (GAD67 vs undiff: *≤0.05; GAT1: vs undiff ***≤0.001). C, D: Western blot with β-actin as loading control was used for quantification of proteins. (*≤0.05 vs undiff). GAD67 and GAT-1 were detected using specific antibodies. (E): DAPI (blue) was used as nuclear counter stain for GAD67 containing cells (green). (F): GAD67 (green) was co-localized with GAT1 (red) in the same differentiated cells (yellow overlay color) (G) GAD67 (green) in undifferentiated HiB5 cells; DAPI (blue): cell nuclei.
Figure 5.
Synthesis of GABA and stimulation-induced release from differentiated HiB5 cells.
(A) GABA was detected using a specific antibody against GABA. (B) GABA release was detected in artificial cerebrospinal fluid (aCSF) without KCl (basal) or with 100 mM KCl (stimulation) in the presence of Ca2+. GABA concentrations in aCSF were determined using HPLC. The data represent the mean ± SEM for GABA release from 6 independent culture experiments (***≤0.0004 vs baseline levels in differentiated cells).
Figure 6.
Expression of GAD65, parvalbumin and calbindin in undifferentiated and differentiated HiB5 cells.
Antibodies were used to detect specific proteins (A) GAD65 (green) (B) parvalbumin (green, right) (C) expression of calbindin (red). Blue: cell nuclei counter stained with DAPI.
Figure 7.
Co-localization of epigenetic regulators HDAC1 and DAXX, developmental transcription factors PAX5 and Runx2 with GAD67 in differentiated HiB5 cells.
Specific antibodies were used to detect several proteins expressed by the target genes associated with a GAD67 regulatory network. GAD67 (green) was co-localized with (A) HDAC1 (red); (B) DAXX (red) and GABA (green) with (C) PAX5 and (D) Runx2 (red fluorescence, respectively).
Figure 8.
Co-localization of GAD67 with (A) GluR5 and (B) expression glutamate receptor subunits GluR6/7 in differentiated cells.
Specific antibodies were used to detect glutamate receptor subunits. (A) GluR5 expression in differentiated HiB5 cells; (B) GluR6/7 expression in differentiated HiB5 cells. GluRs (red) was co-localized with GAD67 (green).