第828回千葉医学会例会・第6回磯野外科例会 88-3.

Western blotting was performed to examine the protein levels of Twist in the indicated cells; β-actin was used as control. (JPG 151 kb

Decreased H4R expression in GCs.

<p>(A) Representative blots of HRH4 expression in normal mucosa and gastric tumor tissues. α-tubulin was used as a stable endogenous control. (B) The histogram shows the anlysis of the results from the immunoblottings. The relative expression value of HRH4 protein (normalized by α-tubulin) in GCs is expressed as an average ratio±s.e. of tumor dose to matched adjacent normal tissue dose. *p<0.01 vs. Control, ANT. (C) Real-time PCR assay was carried out as described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031207#s2" target="_blank">Materials and Methods</a> section, boxplots of relative HRH4 mRNA(HRH4/GAPDH) measured with real-time PCR analysis showing median; box: 25th–75th percentile; bars: largest and smallest values within 1.5 box lengths; little cross: outliers. The results were obtained from 3 reactions in each sample. (D) Representative immunofluorescent microscope analysis of paired samples of GC tissue and adjacent normal tissue using anti human HRH4 monoclonal antibody (red). Nuclei were stain with DAPI (blue). Sample I: gastric cancer (GC); sample II: adjacent normal control (ANT). Arrows point to region of positive staining.</p

Copy number loss of HRH4 in GCs.

<p>(A) Real-time PCR assay was carried out as described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031207#s2" target="_blank">Materials and Methods</a> section, and the results were obtained from indicated group of samples. Boxplots of relative copy number of HRH4 mRNA measured with Real-Time PCR analysis showing median; box: 25th–75th percentile; bars: largest and smallest values within 1.5 box lengths; little cross: outliers. mRNA expression level of HRH4 in groups with deleted (n = 23) or unaltered (n = 108) DNA copies. (B) Representative figure of FISH analysis using chromosome 18q specific alpha satellite DNA probe and chromosome 18q11 specific probe for HRH4 gene. I. Nucleus of ANT tissue with two signals for each of green and orange, showing no deletion of chromosome 18q or HRH4 gene. II. Nucleus of GC tissue with 0–2 signals for green and 0–2 signals for orange, indicating relative deletion in chromosome 18q or HRH4 gene.</p

HRH4 activation induced growth arrest in gastric carcinoma cell lines.

<p>(A) Mock-AGS and H4R-AGS cells were treated with 10⁻⁵ M histamine, CB or CB accompanied by HRH4 antagonist (JNJ7777120) pretreatment, and cell-cycle distributions were determined by propidium iodide flow cytometry analysis. Each value is the mean±s.e. of triplicate data representative for three independent experiments. *p<0.05 and **p<0.01 vs. Control, H4R-AGS cells without any treatment. (B) Colony-formation assay. 5×10³ Mock-AGS and H4R-AGS cells were treated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031207#pone-0031207-g003" target="_blank">Fig. 3A</a> and maintained in G418 for 14 days, and the colonies were stained with Giemsa. The bar graph shows the absolute colony (≥50 cells) number±s.e. in duplicate experiments. *p<0.05 and **p<0.01 vs. Control, H4R-AGS cells without any treatment. (C) Mock-AGS and H4R-AGS cells were treated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031207#pone-0031207-g003" target="_blank">Fig. 3A</a>, WST-1 (Roche) assay measuring the activity of mitochondrial dehydrogenases was performed following the manufacturer's instruction at 0-, 1-, 2-, 3- ,4- ,5- day time points. Each value is the means.e. of triplicate data representative for three independent experiments. Error bars represent standard deviation of the mean.</p

Expressions of cell cycle proteins regulated by HRH4.

<p>Mock-AGS and H4R-AGS were treated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031207#pone-0031207-g003" target="_blank">Fig. 3A</a>, and total cell lysates were examined by western blotting.</p

CNVs of HRH4 in GC tissues and matched adjacent normal tissues (ANTs).

<p>CNVs of HRH4 in GC tissues and matched adjacent normal tissues (ANTs).</p

Comparison of CNVs of HRH4 gene between adjacent normal tissues (ANT) and healthy normal controls (HNC) ^* from peripheral blood.

<p>*ANT represents adjacent normal tissues; HNC represents healthy normal controls</p

FISH results in 10 GC/ANT pairs.

<p>FISH results in 10 GC/ANT pairs.</p

Additional file 3: Figure S2. of Long non-coding RNA MALAT-1 modulates metastatic potential of tongue squamous cell carcinomas partially through the regulation of small proline rich proteins

Establishment of the SCC metastases animal model in nude mice; grossly obvious tumors and metastases were dissected and fixed immediately with 4 % paraformaldehyde for pathological analysis. (JPG 1505 kb