Figure 3. Expression of the mammalian tumor suppressor ARF in zebrafish driven by heat shock promoter. .

In vivo analysis of transgenic zebrafish expressing human ARF under the control of an inducible heat shock promoter, Tg (hsp70l:ARF) (hs:ARF). Schematic of the hs:ARF transgene (top left). The ARF cassette included in the transgene is a cDNA that consists of human exons 1b, 2, and 3 of CDKN2A. Representative Western blot of 3 replicates of ARF before (0 hr) and 3 and 6 hr post heat shock induction of ARF expression (top middle). Portion of fin shown for analysis of expression in vivo (top right; dashed box). Scale bar: 1 mm. Immunostaining (sagittal confocal images) for ARF in adult hs:ARF zebrafish fins at 0, 3, and 8 hr after a single, hour long, 37°C heat shock (bottom). Scale bars: 50 μm. ARF expression is maximal at 3 hr post heat shock, and it is undetectable by 8 hr post heat shock. Figure supplement 1 shows in vitro assays.

DOI:http://dx.doi.org/10.7554/eLife.07702.009

Figure 3—figure supplement 1. Analysis of ARF expression in zebrafish cells.

(A) Immunofluorescence for Mdm2 and ARF (top) and Tp53 (bottom) in zebrafish cells (ZKS) transfected with pcDNA-ARF. ARF and Mdm2 co-localize in the nucleolus (arrow) when ARF is expressed; in cells without ARF, Mdm2 has a diffuse nuclear staining pattern (arrow head; top). Tp53 upregulation depends on ARF expression (bottom). Scale bars: 10 μm. (B) Quantification of Tp53 upregulation in zebrafish cells (ZKS) transfected with pcDNA-ARF (N = 100, p<0.01). Results are shown as mean ± standard deviation.

DOI:http://dx.doi.org/10.7554/eLife.07702.010