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Double labeling with the astrocytic marker GFAP, the neuronal marker NeuN or the motor neuron marker ChAT, respectively, confirmed that p53 labeling was present in neurons and astrocytes (Fig. 7g i), with the latter accounting for most p53-positive cells in white matter.
We next analyzed the expression pattern of motor neuron marker, islet1, using in situ hybridization and a transgenic fish line Tg isl1 GFP); glial lineage marker, glial fibrillary acidic protein (gfap) [15], using heterozygous Tg(gfap GFP), and axon marker, acetylated tubulin (AcTub), using immunofluorescence microscopy.
SMI32 neurofilament antibody is a well-established motor neuron marker (40, 41).
Furthermore, immunofluorescent examinations of spinal cord sections revealed GFP-IR motor neurons that also expressed the motor neuron marker choline acetyl transferase (ChAT) in the ventral horns.
Immunostaining for motor neuron marker ISL1/2 as well as western blot analysis for TUBB3 further confirmed that motor neuron differentiation was compromised in Taf9b KO cells.
Surprisingly, DAPT did not affect induction of the motor neuron marker Islet1 (Isl1) (controls n=5/5; DAPT n=5/5; Fig. 2H-I).
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Moreover, the specific motor neuron markers Lhx3, Lhx4, Isl1, Mnx1, and pan-neuronal marker Tubb3 were among the most down-regulated genes in Taf9b KO cells.
(C) Immunofluorescent images of motor neuron markers (ISL1, HB9) and other neural marker (MAP2) at day 12. Nuclei were stained with Hoechst 33342 (blue).
Human nuclear antigen-positive cells did not double label with markers for the mature motor neuron markers ChAT or SMI-32, indicating that 13 days of survival in vivo was insufficient for differentiation of transplanted hMNPs.
We observed a significant reduction in all motor neuron markers in Taf9b KO cells upon differentiation.
ES cells were differentiated as described previously and motor neuron markers Isl1, Mnx1, and Lhx3 were measured by qRT-PCR.
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