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Additional Supporting Information may be found in the online version of this article.

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HEP_24573_sm_suppfig1.tif488KSupporting Figure 1 Using the lowest dose of clobenpropit found to be inhibitory in all CCA cell lines (10 μM), we found that clobenpropit decreased CCA proliferation beginning at 6 hours and up to 48 hours compared to basal treatment. *p < 0.05 versus basal and data are SEM ± 12 experiments.
HEP_24573_sm_suppfig2.tif448KSupporting Figure 2 MTS assays in nonmalignant cell lines revealed that clobenpropit had no effect at doses of 10 μM stimulated for 6 hours, but began to decrease normal cell proliferation at higher doses (25 and 50 μM) and longer times (48 hrs). *p < 0.05 versus basal and data are SEM ± 12 experiments.
HEP_24573_sm_suppfig3.tif314KSupporting Figure 3 Using the Pearson's Correlation Coefficient analysis in several of our CCA cell lines, we found that the expression of H3HR does not contribute to the cell viability (or cell proliferation) seen after clobenpropit treatment and no significant correlation was detected. Further, using the same analysis we have assessed the correlation between H4HR expression and cell proliferation. Here we found no significant correlation between histamine receptor gene expression and the anti-proliferative effects of clobenpropit.
HEP_24573_sm_suppfig4.tif424KSupporting Figure 4 (a) siRNA transfection resulted in the genetic knockdown of H3HR in Mz-ChA-1 cells at a 50% efficiency rate as shown by real-time PCR for H3HR gene expression. (b) PCNA proliferation by immunoblots in Mz-ChA-1 cells stimulated with clobenpropit in both mock- and H3HR-siRNA transfected cells was performed. The H3HR agonist, RAMH was added as a positive control. In mock-transfected cells, RAMH and clobenpropit significantly decreased CCA growth. In Mz-ChA-1 cells transiently silenced for the H3HR by siRNA (40 nM), clobenpropit significantly inhibited CCA growth cells to a greater extent than in Mz-ChA-1 cells with normal H3HR expression. (c) Overexpression of H4HR was performed in Mz-ChA-1 cells treated with and without clobenpropit (10 μM). Gene and protein expression for H4HR was significantly increased after H4HR transfection compared to LacZ controls. (d) By PCNA protein expression, overexpression of H4HR (2.5 μg) significantly decreased Mz-ChA-1 proliferation compared to LacZ controls. *p < 0.05 versus basal treatment (or scrambled siRNA or LacZ controls) and data are SEM ± 6 experiments for immunoblots and SEM ± 6 for PCR.
HEP_24573_sm_suppfig5.tif248KSupporting Figure 5 Intracellular cAMP and IP3 signaling mechanisms were evaluated in Mz-ChA-1 cells stimulated with clobenpropit (10 μM). By RIA we found that clobenpropit (10 μM) significantly increased intracellular IP3 levels (a), but had no significant impact on cAMP levels (b). *p < 0.05 versus basal treatment and data are SEM ± 12 experiments. (c) MTS assays in Mz-ChA-1 cells stimulated with clobenpropit (10 μM) in the absence or presence of BAPTA/AM, Gö6976 or RpcAMPs demonstrated that BAPTA/AM and Gö6976, but not RpcAMPs block clobenpropit-induced CCA inhibition. *p < 0.005 versus basal treatment and #p < 0.05 versus clobenpropit treatment. Data are SEM ± 12 experiments.
HEP_24573_sm_suppfig6.tif234KSupporting Figure 6 By real-time PCR, clobenpropit significantly decreased the gene expression of MMP -1, -2, -3, -9 and -11 in CCA compared to basal treatment. *p < 0.05 versus basal mRNA and data are SEM ± 3 experiments.
HEP_24573_sm_suppfig7.tif949KSupporting Figure 7 Alamar blue (top) and TUNEL staining (bottom) were used to detect cell viability and apoptosis in Mz-CHA-1 cells cultured in matrigel. There was no difference in cell viability or in the number of apoptotic cells in the matrigel between the basal- and clobenpropit-treated group with either alamar blue assay (top) or TUNEL staining (bottom, viable cells are labeled by red, apoptotic cells are labeled by blue). Alamar blue fluorescence was recorded using a fluorescence microplate reader using 560/590 nm ex/em filter settings, not significant p > 0.05 relative to basal-treatment. TUNEL was evaluated by BrdU incorporation by terminal deoxynucleotidyl transferase at the site of DNA fragmentation and detected by a highly specific and sensitive biotinylated anti-BrdU antibody and visualized by a streptavidin-horseradish peroxidase conjugate. The cells were counterstained with methyl green.
HEP_24573_sm_suppinfo.doc161KSupporting Information.
HEP_24573_sm_supptable1.doc44KSupporting Table 1.

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