Huang Et Al, Supplemental Figure Legends

Huang et al, Supplemental Figure Legends

Supplemental Figure S1:Characterization of the Mz-R1 shRNA cell line.

Mz-ChA-1 cells were stably transfected with human R1 repressor shRNA. The resulting clones (Mz-R1 shRNA) were screened for the greatest degree of R1 repressor knockdown compared to the control-transfected cell line (Mz-Neo neg) by real time PCR. Data are expressed as relative R1 repressor expression (average SD) after normalizing to GAPDH levels. (*p<0.05).

Supplemental Figure S2: Characterization of the Mz-MAOA+ cell line.

Mz-ChA-1 cells were stably transfected with a plasmid containing human MAOA cDNA (pCMV6-MAOA; Origene, Rockville, MD) or the empty expression vector (pCMV6). The expression of MAOA was assessed in the resulting cell lines (Mz-MAOA+ and Mz-CMV6) by real time PCR (A) and immunoblotting (B). Real time PCR data are expressed as relative MAOA expression (average  SD) after normalizing to GAPDH levels. (*p<0.05). Mz-MAOA+ cells were further characterized by assessing their ability to secrete serotonin (C). Serotonin levels were assessed in the conditioned media in Mz-pCMV6 using commercially available EIA kits. Data are expressed as serotonin concentration (average  SD; *p<0.05).

Supplemental Figure S3: Mz-MAOA+ cells have reduced invasiveness.

The invasive capacity of Mz-pCMV6 and Mz-MAOA+ cells was assessed using a commercially available invasion assay. Representative images from each cell line are shown. The invasion index was determined as the percentage of invading cells in the invasion chamber compared to the invading cells in the control chambers (D). Data are expressed as average ± SD (*p<0.05).

Supplemental Figure S4:Model of the regulation of MAOA expression in cholangiocarcinoma.

We demonstrated that the MAOA promoter region was hypermethylated at CpGI 28 and that while there was a correlation between the degree of methylation and MAOA expression, the correlation was not so strong as to exclude the possibility of a second independent mechanism for the suppressed MAOA. In parallel, we demonstrated that IL-6 signaling can also suppress MAOA expression via the modulation of the R1 repressor/SP-1 balance, specifically IL-6 signaling is associated with the nuclear extrusion of the positive transcriptional regulator, SP-1 and the nuclear accumulation of the negative regulator R1 repressor. Blocking both the hypermethylation and IL-6-mediated events could restore MAOA expression in a cholangiocarcinoma cell line back to the MAOA levels observed in a non-malignant cholangiocyte cell line. Taken together, our data suggests that therapies designed to increase MAOA expression and/or decrease serotonin and dopamine production, might prove beneficial in the treatment of cholangiocarcinoma.