Increased HDAC activity in medulloblastoma.

<p>A, HDAC activity was measured from wild type and Smo/Smo mice cerebellum and compared with medulloblastoma from Smo/Smo mice. *P = 0.073. **P<0.005. B, HDAC activity was measured from cytoplasmic and nuclear fractions of wild type cerebellum and Smo/Smo medulloblastoma. C, HDAC activity of individual HDAC members were measured after immunoprecipitation with specific antibodies. Bars correspond to mean ± S.E. Other HDAC members (HDAC3, 4, 7) did not exhibit any significant activity above the background.</p

Differentiating CGP cells show a decline of HDAC activity and protein level.

<p>A, Immunoblot of CGP cells differentiating in vitro. CGP cells isolated from P5 wild type mice were plated and harvested at different days of in vitro culture (DIV). Numbers below immunoblots are the intensity normalized to undifferentiated cells. B, HDAC activity of CGP cells differentiated in vitro was measured. Assay was performed three times in triplicate and the representative one is shown. Bars correspond to mean ± S.E. of three independent experiments.</p

Inhibition of HDAC blocks Shh-induced proliferation.

<p>A, HDAC activity was measured from CGP cells treated with increasing concentrations of TSA along with 0.1 µM SAG for 3 days. Bars correspond to mean ± S.E. *P<0.05. B, Proliferation of CGP cells induced by 0.1 µM SAG were measured with increasing concentrations of Tubastatin A or TSA. Data were expressed relative to the proliferation rate of DMSO-treated control cells. Error bars represent mean ± S.E. **P<0.001.</p

Inhibition of HDAC ameliorates hyperplasia of CGP cells during postnatal cerebellum development.

<p>A, Bright field images of paraffin-embedded mice brain sections that were stained with hematoxylin and eosin. Smo/Smo mice were injected with DMSO, TSA or Tubastatin A for two weeks and then dissected brains were fixed and processed for staining. Arrows indicate hyperplasia of CGP cells after DMSO (control) or Tubastatin A treatment and arrowhead indicates the narrow EGL after TSA treatment. Scale bar represents 100 µm. B, Plot showing the length of EGL from DMSO, TSA or Tubastatin A injected mice. Each symbol represents average length of individual mice and mean ± S.E. were shown. *P<0.0001.</p

HDAC protein and mRNA levels in medulloblastoma.

<p>A, Immunoblot of HDACs 1 through 11 in lysates from wild type cerebellum, Smo/Smo cerebellum and medulloblastoma. Numbers below immunoblots are the intensity normalized to αTubulin. B, Relative quantity of individual HDAC members were measured by qRT-PCR from wild type cerebellum (WT), Smo/Smo cerebellum (Smo CB), and Smo/Smo medulloblastoma (Smo MB) (n = 7). Bars correspond to mean ± S.E. *P<0.05. **P<0.0001.</p

Shh induced HDAC activation and proliferation in CGP cells.

<p>A, Phase contrast image of CGP cells incubated with DMSO, 0.1 µM SAG or 1 µM SAG for 6 days. Scale bar represents 100 µm. B, HDAC activity was measured from CGP cells incubated either with DMSO or different concentrations of SAG for 2, 4 and 6 days. Bars correspond to mean ± S.E. C, Phase contrast images of CGP cells from Smo/Smo mice after 1,3,5 and 7 days in vitro culture. Scale bar represents 100 µm. D, HDAC activity was measured from cultured CGP cells isolated from wild type and Smo/Smo mice. Bars correspond to mean ± S.E.</p