|
Fig. S1
DNA hypomethylation is specific to UHRF1-GFP High larvae. (Related to Figure 1)
(A) Three independent F1 zebrafish lines over-expressing UHRF1 at different levels were generated. Live images (left) and confocal sections through the liver (right) stained with Cy3-SA (red) and DAPI (blue) from 5 dpf transgenic fish confirm the nuclear localization of the transgene and reveal variable levels of expression. With increasing levels of UHRF1-GFP, nuclei become larger and DNA becomes punctate. (B) Endogenous (zebrafish) uhrf1 and transgenic (human) UHRF1 mRNA levels in the liver of each transgenic and control line (nlsmCherry) was determined by qPCR on 5 dpf. Four separate clutches of pooled livers were analyzed per line. Boxes represent 75th and 25th percentile, bars indicate lowest and highest values. (C) Global DNA methylation determined fromslot blots of genomic DNA from the liver of 5 dpf controls (Tg(fabp10:nls-mCherry or non-transgenics)) that were either untreated or treated with 50 μM 5-Aza (n=1) and from Tg(fabp10:UHRF1-GFP) larvae expressing Low, Medium and High levels of UHRF1 in the liver (n=3). Samples blotted in parallel were stained with either methylene blue for total DNA or probed with anti-5MeC. The 5MeC signal was normalized to methylene blue staining. Average, normalized values were divided by the average value in control (untreated livers). p value was determined by Student’s T-test; not significant (n.s.) Error bars are SD.
Reprinted from Cancer Cell, 25(2), Mudbhary, R., Hoshida, Y., Chernyavskaya, Y., Jacob, V., Villanueva, A., Fiel, M.I., Chen, X., Kojima, K., Thung, S., Bronson, R.T., Lachenmayer, A., Revill, K., Alsinet, C., Sachidanandam, R., Desai, A., SenBanerjee, S., Ukomadu, C., Llovet, J.M., and Sadler, K.C., UHRF1 overexpression drives DNA hypomethylation and hepatocellular carcinoma, 196-209, Copyright (2014) with permission from Elsevier. Full text @ Cancer Cell