Figure 6

Dnmt1 and Uhrf1 protein expression in Lsh^−/−cells and activation of intracisternal A-particle protein in DNA hypomethylated cell types. (a) Indirect immunofluorescence on WT fibroblasts using antibodies against Lsh and Dnmt1. Green arrowheads indicate co-localisation. Merge: Lsh and Dnmt1 pseudo-coloured red and green respectively. (b) Indirect immunofluorescence Lsh^−/− fibroblasts as in (a). Crescent-shaped disrupted Dnmt1 foci (yellow arrowheads). (c) Indirect immunofluorescence of WT fibroblasts using Uhrf1and Dnmt1 antibodies. Co-localisation of Uhrf1 and Dnmt1 (green arrowheads). Merge: Uhrf1 and Dnmt1 are pseudo-coloured red and green respectively. (d) Indirect immunofluorescence as in (c). Co-localisation between Uhrf1 and Dnmt1 (green arrowheads). In the merged panel, Uhrf1 is pseudo-coloured red and Dnmt1 is pseudo-coloured green. DNA is stained with DAPI. Scale bar = 5 μM. (e) Lanes: WT, Lsh^−/−: western blots on WT and Lsh^−/− lysates probed with an IAP-gag specific antibody. Lanes: p53^−/−, DN: western blots on p53^−/− and DN lysates probed with the IAP-gag specific antibody. Molecular weight of proteins is indicated in kDa on the right; prominent bands are annotated on the left. Higher mobility protein species (unannotated arrows) may represent IAP gap-pro or gag-pro-pol fusions. Lysate loading control: alpha-tubulin. (f) Indirect immunofluorescence on fixed cells using the same IAP-gag antibody as in (a). Left: WT and Lsh^−/− fixed cells. Right: p53^−/− and DN fixed cells. IAP staining was detected with an Alexa Fluor 488 secondary antibody yielding a green stain. Nuclei are counterstained blue with DAPI. Scale bar = 10 μM. (g) Transmission electron microscopy on Lsh^−/− and DN fixed cells. Left panel: lower magnification of subcellular structures; note the presence of multiple darker round structures. Boxed area is shown at higher magnification on the right. White arrows indicate examples of VLP. Hashed yellow area indicates endoplasmic reticulum cisternae. Scale bar = 500 nM (left) and 100 nM (right). DN; p53−/− | Dnmt1−/− double knockout MEFs.