Fig. 2

Tau localises with TIP5 and impacts on rDNA transcription and heterochromatin. Immunoprecipitation from whole cell lysates showed that tau associates with TIP5 in both undifferentiated (U.SHSY5Y) and differentiated cells (D.SHSY5Y) (ai). Double immunogold labelling revealed that Tau (15 nm) (white arrow) and TIP5 (5 nm) (black arrow) closely associate inside the nucleolus (blue) in SHSY5Y cells (see insert highlighted by black box) (aii). b Western blotting (i) and qPCR (ii) to confirm siRNA tau knockdown in undifferentiated SHSY5Y cells. ci qPCR on samples from the knockdown cells showed a significant increase in 45S-pre-rRNA synthesis (rDNA transcription), 18S rRNA and 28S rRNA processing. [45S pre-rRNA P = 0.017], [18S rRNA P = 0.018]; [28S rRNA P = 0.0038]. (cii) Western blotting shows that proteins levels of TIP5 and UBF are unchanged in tau knockdown cells. d & e Representative immunofluorescence fluorescence images showing labelling for H3K9me2/H3K9me3 control and in knockdown cells. Graphs showing quantification from four independent experiments, each with five images and each containing an average of 30 cells. Quantitative immunofluorescence labelling showed that the tau knockdown resulted in a significant reduction in the levels of H3K9me2 [P < 0.0001] (D) and number of H3K9me3 foci [P < 0.0001] (e). Labelling for 5-Methylcytosine (5-MC) showed that the tau knockdown resulted in a significant reduction in the nuclear levels of 5-mC methylation [P < 0.0001] (f). Analysis of HpaII resistance assay showed that tau knockdown reduces the T0 element methylation (g). *P < 0.05. Experiment Aii = N2. All other experiments N ≥ 4