Expression of the methylcytosine dioxygenase ten-eleven translocation-2 and connexin 43 in inflammatory bowel disease and colorectal cancer

Figure 1 Characterization of the in vitro HT-29 cell model. A: Western blot of connexin 43 (Cx43) protein expression in parental HT-29, HT-29 Cx43-Dendra (Cx43D), and in HT-29 Cx43^- cells. Bar graphs display mean densitometric analysis (of three independent experiments) after normalizing protein expression to glyceraldehyde 3-phosphate dehydrogenase (GAPDH); B: Immunofluorescence micrographs of parental HT-29 and HT-29 Cx43^- cells, as well as a fluorescent micrograph of HT-29 cells transduced with the GFP-Cx43 construct. Highly GFP positive cells were sorted using BD-Fluorescence-Activated Cell Sorting cell sorter, generating the HT-29 Cx43D. Scale bar 10 μm; C: Cells were counted at three different time points (24, 48 and 72 h), using the trypan blue dye exclusion assay. Compared to parental HT-29 cells, HT-29 Cx43D cells had a slower proliferation rate and HT-29 Cx43^- cells a faster proliferation rate at 48 and 72 h; D: Histograms show normalized gene expression of ten-eleven translocation (TETs) in parental HT-29, HT-29 Cx43D and HT-29 Cx43^- cells, as detected by quantitative polymerase chain reaction (qPCR). TET-2 was the highest expressed TET in HT-29 cells, and cells with differential Cx43 expression also had significantly different TET-2 expression levels. The inset is an immunomicrograph of TET-2 in HT-29 C43D cells. Scale bar 5 μm. Results are presented as means ± SEM of three independent qPCR runs in duplicates. One-way ANOVA, ^aP < 0.05; ^bP < 0.001; ^cP < 0.0005. Cx43: Connexin 43; GAPDH: Glyceraldehyde 3-phosphate dehydrogenase.