HRH4 activation induced growth arrest in colorectal carcinoma cell lines. (A) Total cell lysates of wild type Lovo, Mock-Lovo and H4R-Lovo cells were examined by Western blotting. (B) Mock-Lovo and H4R-Lovo cells were treated with 10-5M histamine, clozapine or clozapine accompanied by HRH4 antagonist pretreatment, and cell-cycle distributions were determined by propidium iodide flow cytometry analysis. *p < 0.05 and **p < 0.01 vs. Control, H4R-Lovo cells without any treatment. (C) Colony-formation assay. 5×103 Mock-Lovo and H4R-Lovo cells were treated as in Fig.2B 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. (D) H4R-Lovo cells were treated as in Fig.2B, 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. Error bars represent standard deviation of the mean.