Induction of cell apoptosis by silvestrol.

<p>PLC/PRF/5 HCC cells were seeded onto 4-well chamber slide (25,000 cells/well) and were incubated with 100 nM silvestrol for 24 hours. Apoptotic cells were detected by fluorescence microscopy after DAPI staining. The proportion of cells showing morphological features of apoptosis were counted. *, <i>p</i> < 0.05, t-test.</p

Estimation of tumor growth by bioluminescence.

<p><i>A</i>, <i>In </i><i>vitro</i> bioluminescence imaging of PLC-<i>luc</i> cells. Cells were counted and seeded on black 96-well plates. D-luciferin (150 µg/ml) was added to each well and imaging was performed using IVIS. Bioluminescence is plotted against cell number. <i>B</i>, <i>In </i><i>vivo</i> bioluminescence imaging. Orthotopic tumors were established by direct intra-hepatic injection of PLC-<i>luc</i> cells. One million PLC-<i>luc</i> cells were injected into the left lobe of the liver. The growth of tumors were monitored by bioluminescent imaging using the IVIS. C, The relation between the tumor volume and bioluminescence. After at least 4 weeks of monitoring mice were sacrificed and liver tumor volumes were obtained using caliper measurement. The estimated volume of tumor after excision is plotted against bioluminescence determined in situ.</p

Decrease of mitochondrial membrane potential by silvestrol.

<p>A, PLC/PRF/5 cells were seeded on 4-well chamber slide (25,000 cells/well) and incubated with 100 nM silvestrol for 24 hours. Cells were stained with JC-1 and mitochondrial permeability was detected using fluorescence microscopy. Cells exhibit red fluorescence under basal conditions, but green fluorescence following alteration in mitochondrial membrane potential. B, PLC/PRF/5 cells were seeded on 96-well plate (5000 cells/well) and incubated with 100 nM silvestrol for 24 hours. Cells were stained with JC-1 and the ratio of green to red fluorescence was determined fluorometrically (mean ± SD). *, <i>p</i> < 0.05, t-test.</p

Modulation of expression of apoptosis-related proteins by silvestrol.

<p>(A) PLC/PRF/5 HCC cells were seeded on 96-well plate (5,000 cells/well) and incubated with 100 nM silvestrol for up to 24 hours. Caspase-3/7 activation was assessed using a luminometric assay (Caspase-Glo 3/7 Assay, Promega Corp., Madison WI). The data represents the mean and SD of three separate determinations. (B) PLC/PRF/5 cells were seeded on 6-well plates (20,000 cells/well) and incubated with 100 nM silvestrol (100 nM) for up to 24 hours. Cells were harvested at the indicated times, and immunoblot analysis for apoptosis-related proteins was performed. *, <i>p</i> < 0.05, ANOVA, Fisher’s PLSD (C) PLC/PRF/5 cells and HepG2 cells were seeded on 6-well plates and incubated with 100 nM silvestrol for up to 24 hours. Expression of Mcl-1 protein was assessed by immunoblotting. (D) RNA was extracted at each time point and Mcl-1 mRNA expression level was assessed by quantitative real-time PCR. Values are expressed relative to expression in no-treatment control after normalization using GAPDH as an internal control. The data represents the mean and SD. *, <i>p</i> < 0.05, ANOVA, Fisher’s PLSD.</p

Cytotoxicity of silvestrol on HCC cells.

<p>Human HCC cells were seeded onto 96-well plates (5,000 cells/well) and incubated with various concentrations of silvestrol or control (diluent). Cell viability was assessed after 72 hours using a metabolic live cell assay (CellTiter 96 AQ, Promega Corp., Madison, WI). The data represents the mean and SD of five separate determinations.</p

Synergistic effect between silvestrol (SVL) and sorafenib (SRF) or rapamycin (RPM).

<p>PLC/PRF/5 cells were seeded on 96-well plates (5000 cells/well) and were incubated for 72 hours with various concentrations of silvestrol (SVL) and (A) sorafenib (SRF) at a fixed ratio of SVL:SRF of 1:250 or (B) rapamycin (RPM) at a fixed ratio of SVL:RPM of 1:2.5. Cell viability was assessed using a metabolic assay (CellTiter 96 AQ, Promega Corp., Madison, WI). Potential interactions between silvestrol and sorafenib or rapamycin were evaluated using the median effects analysis of Chou and Talalay to derive the (C) combination index and (D) dose-reduction index of the combinations.</p

Loss of NAC1 Expression Is Associated with Defective Bony Patterning in the Murine Vertebral Axis

<div><p>NAC1 encoded by <i>NACC1</i> is a member of the BTB/POZ family of proteins and participates in several pathobiological processes. However, its function during tissue development has not been elucidated. In this study, we compared homozygous null mutant Nacc1^-/- and wild type Nacc1^+/+ mice to determine the consequences of diminished NAC1 expression. The most remarkable change in Nacc1^-/- mice was a vertebral patterning defect in which most knockout animals exhibited a morphological transformation of the sixth lumbar vertebra (L6) into a sacral identity; thus, the total number of pre-sacral vertebrae was decreased by one (to 25) in Nacc1^-/- mice. Heterozygous Nacc1^+/- mice had an increased tendency to adopt an intermediate phenotype in which L6 underwent partial sacralization. Nacc1^-/- mice also exhibited non-closure of the dorsal aspects of thoracic vertebrae T10-T12. Chondrocytes from Nacc1^+/+ mice expressed abundant NAC1 while Nacc1^-/- chondrocytes had undetectable levels. Loss of NAC1 in Nacc1^-/- mice was associated with significantly reduced chondrocyte migratory potential as well as decreased expression of matrilin-3 and matrilin-4, two cartilage-associated extracellular matrix proteins with roles in the development and homeostasis of cartilage and bone. These data suggest that NAC1 participates in the motility and differentiation of developing chondrocytes and cartilaginous tissues, and its expression is necessary to maintain normal axial patterning of murine skeleton.</p> </div

Strategy used in generation of <i>Nacc1</i> knockout allele.

<p><b>A</b>. Schematic illustration of gene targeting and Southern strategies. Black lines and blue lines indicate the expected sizes of fragments detected by Southern hybridization of genomic DNAs digested by HindIII/EcoRV and XbaI, respectively. The black bar indicates the probe for Southern hybridization. The fragments within the square on the top are the expected bands from cross-hybridization of the probe to the genomic DNA in Chr. 6. Note <i>Nacc1</i> is located in Chr. 8. <b>B</b>. Representative clones from Southern hybridization as shown in panel A. Two positive clones, c72 and c156, were identified. Clones c73 and c74 are partially targeted because they lack the first LoxP site as shown in panel A. <b>C</b>. Quantitative real-time PCR to measure neo levels revealed that both positive clones, c72 and c156, likely have a single copy insertion of the neo cassette. The housekeeping gene control used in this experiment was <i>Rpp30</i>, and the data was normalized to single neo copy controls (T/O +/- and TET2 k-in) to calculate relative neo levels.</p

Reduced NAC1 expression is associated with alterations in chondrocytic expression of <i>matrilin-3</i> and <i>matrilin-4</i>.

<p>Quantitative real-time PCR analysis performed on cultured costal chondrocytes derived from Nacc1^+/+ (wild type) and Nacc1^-/- (knockout) mouse pups at postnatal day 1 (P1) to measure the relative expression levels of the different matrilin (MATN) proteins. Only MATN3 and MATN4 are expressed at a significantly different level (** denotes p < 0.01) between the two genotypes.</p

NAC1 expression in the chondrocytes of wild type mouse embryos.

<p>Comparison of representative para-sagittal sections of Nacc1^+/+ (wild type, <b>A</b>), Nacc1^+/- (heterozygous, <b>B</b>), and Nacc1^-/- (knockout, <b>C</b>) mouse embryos at embryonic day 16 (E16) shows the widespread distribution of NAC1 in various tissues, including chondrocytes (boxed regions). The wild type expression pattern exhibits widespread immunoreactivity including such axial tissues as vertebral primordia and the peri-ventricular germinal zones in the brain. <b>D</b>. Chondrocytes in cartilaginous tissues of a human embryo (estimated gestational age, 7 weeks) also exhibit extensive NAC1 immunoreactivity. Anti-NAC1 immunohistochemistry (using species-specific primary antibodies) with hematoxylin as the counterstain.</p