Milea Timbergen

102 and FOXK2 in T41A samples was downregulated suggesting that the hypermethylation observed in T41A samples reduces mRNA expression. Table 3. Expression levels of genes identified by DMRs between S45F and T41A DTF samples. Transcript levels are derived from publicly available Affymetrix based mRNA expression data (GSE58697) of 45 T41A and 34 S45F tumours.. Some genes are represented by multiple probe sets. Mann-Whitney U test was used to assess statistical significance in transcript levels of the respective genes between S45F and T41A tumours. Gene name Probe number Median (IQR) p-value CCDC6 204716_at 203 (126-239) 0.034 225010_at 617 (554.6-703.4) 0.579 FOXK2 242937_at 17 (15-21) 0.004 242938_s_at 34 (32-39) 0.384 226224_at 149 (141-158) 0.373 203064_s_at 112 (101-127) 0.533 DUX4L6 216472_x_at 11 (8-14) 0.362 208201_at 15 (11-20) 0.510 NLRP4 242334_at 2.7 (1.2-4.2) 0.149 NOC4L 218860 50 (44-55) 0.628 PERM1 224501_at 1 (1-1) 0.415 CTNNB1 (β-catenin) – DNMT1 protein interaction. To verify whether the observed DMRs between S45F and T41A DTF samples could be the result of a differential regulatory interaction between β-catenin mutants and DNMT1, an immunoprecipitation experiment was performed. FLAG-tagged wild-type β-catenin and different β-catenin mutants (T41A, S45P, exon 3 deletion mutant, K335I) were transiently expressed in HEK293T and HCT116 cells. Western blot analysis of total lysates of the transfectants demonstrated expression of DNMT1 as well as the FLAG-tagged version of β-catenin and β-actin as loading control (Figure 3, Supplemental Figure 5). When FLAG- tagged β-catenin was immunoprecipitated no DNMT1 was co-precipitated. We tentatively conclude that we cannot verify a protein-protein interaction between β-catenin wild-type or mutants and DNMT1. 4