Fig. 6.

Colony formation assay in the parathyroid cell line sHPT-1 and HEK293T cells. a Transfection of a TET1 expression vector and not the empty vector resulted in increased expression of TET1 at the messenger RNA (mRNA) and protein levels and b increased the global level of 5hmC (lanes 1 and 3, transfection of sHPT-1 cells for 72 h and 10 days, respectively; lanes 2 and 4, empty vector). c Colony formation assay, representative results are shown. Transfected cells were selected by incubation with G418 (neomycin) for 10 days. sHPT-1 cells also appeared as smaller colonies not necessarily apparent in c. d Quantification of triplicates (mean ± SEM); sHPT-1/TET1 = 46 ± 7.1 and sHPT-1/empty vector = 175 ± 14.9. HEK293T/TET1 = 176 ± 5.6 and HEK293T/empty vector = 190.6 ± 13.7. e Effects on apoptosis was analyzed in triplicates by quantifying cytoplasmic histone-associated-DNA-fragments 72 h after transfection and after 10 days of G418 selection. Incubation in 0.1 μg/ml camptothecin for 72 h was used as a positive control. f Flow cytometry analysis of sHPT-1 cells 72 h after transfection and after staining with annexin V-FITC and propidium iodide. Early apoptotic cell population in the upper left quadrant and late apoptotic cells in the upper right quadrant. Alive and dead cell populations in the lower left and right quadrant, respectively. Incubation in 0.1 μg/ml camptothecin for 4 h was used as a positive control (data not shown)