Fig 2: Rb1 hyperphosphorylation and E2f1 binding of the human ARF promoter in the blastema during regeneration.(A) Representative Western blot of three experimental replicates of Rb pathway components, E2f1, Rb1, and hyperphosphorylated Rb1 (p-Rb1), before injury (uninj.), at 2 dpa, and during embryogenesis at 72 hpf (left). Quantification of p-Rb1 and Rb1 levels normalized to β-Actin and relative to uninjured tissue. Results are from three independent biological replicate experiments and are shown as mean ratios ± standard deviation. *p<0.05; ***p<0.001 (right). (B) Confocal images of coronal vibratome sections immunostained for Green fluorescent protein (GFP), Msxb, and p-Rb1 in uninjured and regenerating (2 dpa) ARF:GFP fins. Scale bars: 50 μm. Very little p-Rb1 staining is seen in the uninjured fin, but high levels of p-Rb1 staining can be seen in Msxb + cells in the blastema at 2 dpa. The white dashed line represents the amputation plane. (C) Representative ChIP qPCR data of three experimental replicates with a pool of 30 fins per experiment. Tissue was collected from ARF:GFP transgenic fish before injury (uninj.), at 2 dpa (regenerate only), and at 72 hpf. Fold enrichment of E2f1 binding was normalized to rabbit IgG. The zebrafish thymidine kinase 1 (tk1) promoter was used as a positive control for E2f1 binding. Sequences 2 kbp upstream of tk1 were used as a negative control (tk1-). Values above twofold (black dashed line) are significant (p<0.05). Figure supplement 1 shows promoter sequences for the ARF, tk1, and tk1- promoters annotated for canonical E2f binding sites. hpa: Hours postamputation.DOI:http://dx.doi.org/10.7554/eLife.07702.007

Fig 3: Linc00441 could suppress RB1 expression(A) and (B) The relative expression of RB1 in cells treating with Linc00441 overexpression or shRNA lentivirus. (C) The protein expression of RB1 and phosphorylated RB1 in cells treating with Linc00441 overexpression or shRNA lentivirus. (D) Relative RIP experiments were performed using an antibody against RB1 on extracts from AGS with IgG as a negative control. The enrichment of the Linc00441 was normalized to the input. The purified RNA was used for RT-PCR analysis. The results showed that no bands were detected from the RNA in the group with anti-RB1.

Fig 4: Model of ARF function in the context of Rb pathway activity during zebrafish development and fin regeneration.ARF is not active during development during which a moderate level of mitogenic signaling causes modest phosphorylation of Rb1 (top); however, during regeneration, high mitogenic signaling induces Rb1 hyperphosphorylation and abundant free E2f1, which activates ARF and leads to inhibition of regeneration (bottom). The dashed lines represent the amputation plane.DOI:http://dx.doi.org/10.7554/eLife.07702.019

Fig 5: Cdkn2b inactivation is indispensable for Rb phosphorylation of ductal cells. (a) Immunohistochemistry staining for total Rb and Rb phosphorylation at S780 was conducted on fresh adjacent sections from pancreases infected with the pTomo empty vector, KRAS-shp53-EGFP, KRAS-shp53-EGFP-shCdkn2a, KRAS-shp53-EGFP-shCdkn2b or KRAS-shP53-EGFP-shCdkn2a/b lentiviral particles. Scale bars: 20 μm. (b) The ratio of Rb phosphorylation-positive cell to EGFP-positive cells in a. (c) Immunohistochemistry staining for Rb1 phosphorylation in mouse pancreatic cancer. Scale bars: 40 μm. The quantification value is presented as the mean±s.e.m. ***P<0.001.