Research Paper Volume 7, Issue 11 pp 911—927

A novel autosomal recessive TERT T1129P mutation in a dyskeratosis congenita family leads to cellular senescence and loss of CD34+ hematopoietic stem cells not reversible by mTOR-inhibition


Figure 2. Analysis of nuclear clustering of the TERT T1129P mutation in a cell culture model. (A) Representative confocal images of transiently co-transfected HeLa-cells. HA-TERT or HA-TERT T1129P, respectively, harboring 3 HA-tag sequences in frame at the N-terminus were transfected and fixed 48h later. On the indicated pictures e) and k) equal amounts of the TERC minigene was co-transfected together with the respective TERT minigene. The HA-tag was visualized with a mouse monoclonal anti-HA antibody and an anti-mouse secondary antibody linked to Alexa 488 as described in Materials and Methods. The nucleus was visualized by DAPI staining. The panels on the right represent overlays of the TERT wild type or the T1129P mutations, respectively, with the nucleus stained with DAPI. Cells depicted represent the subcellular distribution pattern seen in >90% of the transfected cells. (B) and (C) Quantification of nuclear accumulation and clustering. For quantification of nuclear accumulation and clustering in the nuclei, 100 cells each from 3 independent transfections have been assessed and counted visually for the presence of nuclear staining and/or nuclear clustering. For statistical analysis a student's unpaired two-tailed t-test was used.