Fig. 2: Hypermethylation of the SatIII DNA locus by D-TALES diminishes SatIII RNA expression and increases sensitivity towards etoposide.

A Schematic representation of SatIII DNA locus with all CpG localizations (red) as well as the TALE-binding sites (green). B Representative images of the co-localization of TALE-GFP (green) and SatIII RNA (red). HeLa cells were transfected with the TALE-GFP construct and exposed to HS conditions (1 h at 44 °C) 24 h after transfection in order to induce SatIII RNA expression and foci accumulation. Cells were fixed, immunostained, and imaged. The histogram indicates co-localizations, represented by overlapping peaks of fluorescence intensities. C Methylation level of the SatIII DNA locus in D-TALEa (active) and D-TALEi (inactive vector control) transfected U2OS cells. At 24 h and 48 h post-transfection U2OS cells were exposed to 44 °C for 1 h (HS) conditions and harvested. Cells were FACS-sorted and mCherry positive cells used for DNA extraction and subsequent pyrosequencing of the SatIII DNA locus. Shown is the percent of methylation to a transfection control. D Quantitative PCR analyses for SatIII RNA expression of samples from (C). Samples incubated for 48 h with the transfection mix were used. Error bars represent standard deviation of the mean of two individual replicates. E Drug response of U2OS cells either transfected with D-TALEa or an inactive control plasmid (D-TALEi). Viabilities were examined by AlamarBlue. Error bars represent standard deviation of the mean of three replicates. Two-tailed paired Student’s t test significant P-values are marked: < 0.05 with (*), < 0.01 with (**), < 0.001 with (***). F Caspase-3/-7 assay of U2OS cells either transfected with the D-TALEa plasmid or an inactive control (D-TALEi). At 24 h post-transfection, cells were treated with different concentrations of etoposide or a DMSO control for 24 h. Depicted are the differences of blue fluorescence (BFU) signals between D-TALEa and D-TALEi. Error bars represent standard deviation of the mean of the three replicates. Two-tailed paired Student’s t-test significant P-values are marked: < 0.05 with (*), < 0.01 with (**), < 0.001 with (***). G Cell viability of HCC827 cells stably expressing either shRNA which targets SatIII (shSatIII, blue) or a shGFP-Control (red). Cells were exposed to 44 °C for 1 h (HS) and afterwards immediately treated with the indicated etoposide concentrations. After an additional 48 h cell viability was measured using AlamarBlue.