Fig. 3

UHRF1 Overexpression in Hepatocytes Induces Tp53-Mediated Senescence

(A) Intense senescence-associated β-galactosidase (SA-b-gal) staining was detected in the liver (outlined) of 5 dpf UHRF1-GFP High larvae compared to light or no staining in controls.

(B) Five dpf fish from five clutches were scored for hepatic SA-b-gal staining. ***p < 0.0001 by Fisher’s exact test.

(C) Nuclear size was measured in hepatocytes of a single control or UHRF1-GFP High 5 dpf liver, and cells were stratified according to GFP expression. Inset shows confocal stack of the DNA organized into foci. **p < 0.001 and ***p < 0.0001, compared to nuclear size in nls-mCherry larvae.

(D) BrdU-positive cells and the total number of transgene-expressing hepatocytes in nls-mCherry and UHRF1-GFP High larvae (bottom) 5 dpf larvae. A Fisher’s exact test was used to calculate p value. In nls-mCherry larvae, most BrdU-positive cells also express the transgene, whereas the BrdUpositive cells in UHRF1-GFP High livers did not express GFP (white arrows in magnified regions, which are marked by the white box).

(E) Heatmap of log2 values from RNA-seq shows cell-cycle regulators are down and Tp53 target genes (marked by *) are up in UHRF1-GFP High 5 dpf livers.

(F) tp53 and cdkn1a mRNA expression were induced on 5 dpf and downregulated on 20 dpf in UHRF1-GFP High livers. *p = 0.05; ***p = 0.001 calculated by one sample Student’s t test. Error bars represent SD.

(G) 5-Aza induces Tp53 expression in primary mouse hepatocytes. Student’s t test was used to determine p value with SD indicated by the error bars across three replicates.

(H–K) tp53^+/- in UHRF1-GFP High larvae significantly reduced SA-b-gal staining in the liver (two clutches) (H), and increased the percent of larvae with normal liver size (I), the area of the left liver lobe (J), and survival at 5 dpf (K). p values were calculated with a Fisher’s test with Freeman- Halton extension (H), Fisher’s exact test (I), and Student’s t test. Boxes represent 75th and 25th percentile, horizontal line is the median, and whiskers mark lowest and highest values (J).