Primers used for real-time PCR amplification.

<p>Primers used for real-time PCR amplification.</p

Down-regulation of Gli decreased ovarian cancer cell migration and invasion.

<p>(<b>A</b>) Exposure to GANT61 (30 µM; 48 hr) reduces expression of both Gli1 and Gli2 protein, determined by Western blot. GAPDH was used as the loading control. (<b>B</b>) Expression of both Gli1 and Gli2 mRNA is decreased following treatment with GANT61 (30 µM; 48 hr), determined by real-time PCR. (<b>C, D</b>) GANT61 inhibits expression of Gli2 in ovarian cancer cells. ES2 and SKOV3 cells were treated with control vehicle (DMSO) or GANT61 (20 µM) for 48 hr. Subsequently, cells were stained with Gli2 and visualized under a confocal microscope. Scale bar, 10 µm. (<b>E, F</b>) Blockade of Hh signaling inhibits ovarian cancer cell migration. Confluent ES2 cell monolayers were wounded with a pipette tip and then treated with N-Shh conditional medium plus control vehicle (0.2% DMSO) or GANT61. Cell migration to the wound area was monitored by microscopy. The percentage of total area covered by cells was then assessed using the NIH Image program. (<b>G, H</b>) Blockade of Hh signaling inhibits ovarian cancer cell invasion. SKOV3 cells (1×10⁵ cells/well) were seeded in the upper chamber. Growth medium containing N-Shh (0.5 µg/ml) alone or together with GANT61 (20 µM), control vehicle (0.2% DMSO) were added to the lower chamber. After 24 hr of incubation, cells that invaded the lower surface of the insert were stained with crystal violet and counted by microscopy. The representative images from three independent experiments are shown. Scale bar, 200 µm. The data are expressed as mean ± SD for experiments performed three times. **, <i>P<</i>0.01, compared with control groups.</p

Gene expression profiles in GANT61-treated SKOV3 cells.

<p>(<b>A</b>) Identification of differentially expressed genes (DEGs) in GANT61-treated SKOV3 cells. Cells were treated with GANT61 (20 µM) or DMSO for 60 hr, and total RNA was extracted as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088386#s4" target="_blank">Methods</a>. Changes in gene expression were determined by cDNA microarray gene profiling using the Illumina Human HT expression BeadChip V4 (Illumina Inc., San Diego, CA). Differential expressions for the DEGs are shown. (<b>B</b>) The top 11 canonical signaling pathways influenced by inhibition of Gli1/Gli2 function in SKOV3 cells. The top 11 canonical signaling pathways, determined by IPA, that were significantly up-regulated or down-regulated by GANT61 treatment in SKOV3 cells, are shown. The 412 DEGs were mapped to the IPA-defined network. The significant <i>P</i>-values that determine the probability that the association between the genes in the dataset and the canonical pathway is by chance alone were calculated by Fisher's exact test, and are expressed as −log (<i>P</i>-value). (<b>C</b>) Heat map of DEGs in the focal adhesion signal pathway in GANT61-treated SKOV3 cells. The heat map shows that 17 genes were significantly differentially expressed, including seven up-regulated genes and 12 down-regulated genes, in the GANT61-treated compared to control SKOV3 cells. (<b>D</b>) Selected DEGs from cDNA array gene expression profiling analyzed by real-time PCR. SKOV3 cells were treated with DMSO or GANT61 for the indicated times, total RNA was extracted for real-time PCR as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088386#s4" target="_blank">Methods</a> using the primer sets in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088386#pone-0088386-t001" target="_blank"><b>Table 1</b></a>. The data represent the mean ± SD of three determinations, and GAPDH was used to normalize the relative mRNA levels.</p

ITGB4-FAK signaling mediates Shh-induced cellular migration and invasion.

<p>(<b>A, B</b>) Hh signaling activates FAK through ITGB4. SKOV3 cells were treated with ITGB4 antibody or control IgG in the presence or absence of N-Shh (0.5 µg/ml) for 24 hr. Expression and phosphorylation of FAK (Tyr397) were verified by Western blot. (<b>C–E</b>) Blockade of Gli down-regulates phosphorylation levels of FAK (Tyr397) but not FAK protein expression. SKOV3 cells were treated with GANT61 or DMSO for 24 hr. Then Western blot was performed using the indicated antibodies. N.S, no significance. (<b>F, G</b>) Repression Gli expression decreases the phosphorylation of FAK (Tyr397) and cytoskeletal organization. SKOV3 cells were treated with GANT61 for 48 hr. Subsequently, cells were stained with antibody against phosphorylation of FAK (Tyr397) and visualized using confocal microscope (<b>F</b>). For F-Actin staining, cells were incubated with Alexa Fluor 488 phalloidin (Invitrogen, A12379) for 20 min and visualized using confocal microscope (<b>G</b>). (<b>H</b>) Loss of Gli-FAK signaling impairs Shh-induced ovarian cancer cell invasion. SKOV3 cells were transfected with control miRNAi or Gli1 miRNAi (miR-Gli1-720) for 24 hr followed by stimulation with N-Shh (0.5 µg/ml) together with PF573228 (5 µM), a specific inhibitor of FAK, or control vehicle (DMSO) for another 24 hr. Cell invasion was measured by invasion assay as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088386#s4" target="_blank">Methods</a>. The data are expressed as mean ± SD for experiments performed three times. **, <i>P<</i>0.01, compared with control groups. Scale bar, 20 µm.</p

Effect of Gli blockade on growth of human ovarian cancer cells <i>in vivo</i>.

<p>SKOV3 cells (2×10⁷) were implanted s.c. into the flank of BALB/c nu/nu mice. Afterwards the mice were received either GANT61 (25 mg/kg s.c., thrice per week) or solvent (corn oil: ethanol, 4∶1). Treatment was initiated in all groups (n = 8 each group) when mean tumor volume had reached 100 mm³. (<b>A</b>) Treatment with GANT61 led to a significant growth inhibition of xenografted human ovarian cancer tumors. n = 8; **, <i>P</i><0.01. (<b>B</b>) The experiment was terminated on day 15, and tumors were excised. Final tumor weights in the GANT61 treatment group (n = 8) were significantly lower compared with excised tumors of the solvent control group (n = 8). **, <i>P</i><0.01. Columns, mean; bars, SD. (<b>C, D</b>) Western blot analyses for Gli1 and Gli2 in tumor tissues. Treatment with GANT61 diminished expression of Gli1 and Gli2. (<b>E, F</b>) Immunohistochemical image of tumor tissue section. Treatment with GANT61 diminished expression of ITGB4 (<b>E</b>). A substantial decrease in phosphorylation of FAK (Tyr397) was also effectively achieved by GANT61 treatment (<b>F</b>). Scale bar, 20 µm.</p

ITGB4 mediates Shh-induced ovarian cancer cell invasion.

<p>(<b>A, B</b>) Stimulation of N-Shh up-regulates ITGB4 expression. SKOV3 cells were incubated in the presence or absence of N-Shh (0.5 µg/ml) for 24 hr. Western blots were then performed for the indicated antibodies. Protein expression was quantified by Image J and normalized to GAPDH. (<b>C, D</b>) Inhibition of Gli1 down-regulates ITGB4 expression. SKOV3 cells were treated with GANT61 or DMSO for 48 hr. Western blot was then performed with the indicated antibodies. Protein expression was quantified by Image J and normalized to GAPDH. (<b>E, F</b>) Blockade of ITGB4 inhibits cell invasion in SKOV3 cells. SKOV3 cells grown in low serum (2% FBS) growth medium were seeded into the upper chambers. The low serum growth medium containing N-Shh (0.5 µg/ml) together with anti-ITGB4 antibody (0.2 µg/ml) or control IgG was added to the lower chamber. After 24 hr of incubation, cells that invaded the lower surface of the insert were stained with crystal violet and counted by microscopy. (<b>G</b>) Gli1 miRNAi inhibits the expression of ITGB4 in SKOV3 cells. SKOV3 cells were transiently transfected with vehicle (lipofectamine alone), control miRNAi and Gli1 miRNAis (miR-Gli1-720 and miR-Gli1-1863), respectively. Western blot showed that Gli1 miRNAi inhibited the expression of ITGB4 genes. (<b>H</b>) Silencing Gli1 expression impairs Shh-induced ovarian cancer cell invasion. SKOV3 cells were transfected with Gli1 miRNAi or control miRNAi for 24 hr followed by stimulation with N-Shh (0.5 µg/ml) or control medium for another 24 hr. Cell invasion was measured as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088386#s4" target="_blank">Methods</a>. (<b>I, J</b>) Silencing ITGB4 expression impairs Shh-induced ovarian cancer cell invasion. SKOV3 cells were transfected with ITGB4 miRNAi constructs (miR-ITGB4-2255 and miR-ITGB4-4027) or control miRNAi for 24 hr followed by stimulation with N-Shh (0.5 µg/ml) or control medium for another 24 hr. Western blot showed that ITGB4 miRNAis inhibited the expression of ITGB4 gene (<b>I</b>). Cell invasion was measured as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088386#s4" target="_blank">Methods</a> (<b>J</b>). The data are expressed as mean ± SD for experiments performed three times. **, <i>P<</i>0.01, compared with control groups.</p