Fig 2: (A) Pedigree of a pair of siblings carrying a heterozygous CDKN2A germline variant concurrently resulting in known missense variants of the two transcripts P16 and P14, NM_000077.4:c.301G>T (NP_000068.1:p.Gly101Trp) and NM_058195.3:c.344G>T (NP_478102.2:p.Arg115Leu). For clarity, this genotype will be hereafter referred to as CDKN2AWT/#. In the diagram, only the amino acid change in P16 (G101W) is indicated for each relative, if known. Age at death is indicated by a d., age of onset of the disease by an o. Individual II-5 developed uterine cancer at a young age and bladder cancer at an older age. III-4 suffers from a carcinoma of the external auditory canal, whereas the cancer entities of I-1 and I-4 were unknown. Consultant probands P1 (IV-1) and P2 (IV-2) are highlighted with an arrow. If the CDKN2A variant was confirmed by genetic analysis, it is displayed in bold. The displayed age denotes the age at which cancer was diagnosed. (B) Schema of iPSC generation by reprogramming of CDKN2AWT/# patient hair keratinocytes and subsequent differentiation to pancreatic progenitors (PPs). (C) Keratinocytes growing out from a hair root and an iPSC colony derived from reprogrammed keratinocytes. (D) IF staining confirmed expression of pluripotency markers OCT4, NANOG, and SSEA4 in the generated patient-specific iPSCs. Scale bar: 50 µm. (E) Sequencing of iPSCs from both donors confirming the heterozygous CDKN2A variant, chr9:g.21971058C>A (hg38). (F) Patient-specific iPSCs differentiated efficiently into PPs. Representative original flow cytometry plots of undifferentiated iPSCs and CDKN2AWT/# P2 iPSCs differentiated for 13 days to PPs are shown next to quantification of PDX1/NKX6-1 double positive cells. n = 3; ordinary one-way ANOVA with Tukey’s multiple comparison test. (G) IF staining for PP marker PDX1 and NKX6-1. Scale bar: 50 µm.

Fig 3: Regulation of p16 and IL6 on the inhibitory effect of sorafenib on cell viability in HCC.a HepG2 and Hep3B cells were transfected with p16 overexpression vector and siRNA targeting p16, respectively. After 24 h, cells were incubated with different concentrations of sorafenib for 24 h. MTT assay was conducted to test cell viability. b After cultured with normal medium (Ctrl) or serum-free medium (Starvation) for 24 h, HepG2 cells were stained with ß-gal solution. In Starvation group, senescent cells that exhibited blue staining were frequently observed under microscope. c p16 expression was detected by western blot. d IL6 concentration in cell supernatant was measured by ELISA. e MTT assay was used to determine the different effects of sorafenib on cell viability in HepG2 with routine culture (control group) or serum depletion (starvation group). f, g After cultured with normal medium (Ctrl) or serum-free medium (Starvation), cells were transfected with siRNA targeting p16 for 24 h (f) or pretreated with IL6 neutralizing antibody (5 ng/ml) for 2 h (g), and then incubated with 10 µM sorafenib for 24 h. MTT assay was conducted to test cell viability. NC: negative control. h In a transwell co-culture system, parent HepG2 cells were seeded in upper chamber, and the cells transfected with pCMV-p16 or empty vector were seeded in the bottom of wells. After 48 h-co-culture, the supernatant in bottom cells was collected to detect IL6 concentration. i The parent HepG2 cells in upper chamber were incubated with different concentrations of sorafenib for 24 h. Then cell viability was tested by MTT. *p < 0.05; **p < 0.01; ***p < 0.001, compared with control (Ctrl). The scale bars represent 25 µm. All immunoblots indicate molecular size markers in kDa

Fig 4: Reducing Adenoviral vector expressing small hairpin RNAs targeting kinesin family member 20B (Ad-shKIF20B) attenuates the proliferation of hepatocellular carcinoma cells in a p53/p14 dependent method. A, Western blots for indicated proteins of HepG2 cells 24 h after receiving adenoviral vector expressing shRNAs targeting KIF20B (Ad-shKIF20B) or pifithrin-a. MOI = 1, pifithrin-a concentration = 400 nmol/L. B, MTT results of HepG2 cells 72 h after indicated treatments. MOI = 1, pifithrin-a concentration = 400 nmol/L, VX-680 concentration = 150 nmol/L for 2 h to induce mitotic slippage. Three independent experiments were carried out. C, Western blots for indicated proteins of Hep3B cells 24 h after receiving Ad-shKIF20B or shp14 transfection. MOI = 1. D, MTT results of Hep3B 72 h after indicated treatments. MOI = 1, VX-680 concentration = 150 nmol/L for 2 h to induce mitotic slippage. Three independent experiments were carried out. (***P < .001)

Fig 5: ARF, not normally present in highly regenerative vertebrates, is specifically activated in blastemas of ARF transgenic zebrafish.(A) Comparison of amino acid sequences of proteins produced by the Cdkn2a/b loci of zebrafish (Danio rerio), amphibians including the axolotl (Ambystoma mexicanum) and western clawed frog (Xenopus tropicalis), chickens (Gallus gallus), and mammals including the mouse (Mus musculus) and human (Homo sapiens). While Cdkn2a and Cdkn2b are conserved and encode Ink4 orthologs, Arf evolved recently and orthologs do not exist in highly regenerative vertebrates including teleost fish and urodele amphibians. (B) Schematic of transgene expressing cytoplasmic Green fluorescent protein (GFP) under the control of the human ARF promoter (top). The promoter consists of human regulatory sequences 736 bp upstream of the transcriptional start site (TSS) of ARF. Immunostaining (wide-field images) for GFP at 24 hpf, 48 hpf, and 72 hpf in wild type (WT) and ARF:GFP embryos (bottom). Scale bars: 200 µm. GFP expression is visible in the hearts of transgenic fish due to presence of a separate transgene used for selection (cmlc2:GFP). (C) Whole-mount in situ hybridization for GFP at 24 hpf, 48 hpf, and 72 hpf in WT and ARF:GFP embryos. Scale bars: 100 µm. Alkaline phosphatase staining is detected in the hearts of transgenic fish (arrow heads) because of the selection transgene as in (B). (D) Confocal images of coronal vibratome sections immunostained for GFP, Msxb, and Proliferating cell nuclear antigen (PCNA) at 2 dpa in WT and ARF:GFP fins. Scale bars: 50 µm. GFP expression is induced in the proliferative blastema of the regenerate, but it is not expressed in the surrounding epithelium. White dashed lines represent amputation planes. (E) GFP intensity (green line) in the regenerates of ARF:GFP transgenic fish relative to WT fish after amputation. The black line represents the percentage of EdU + cells in the regenerates of WT fish after amputation (N=3; secondary axis). Figure supplement 1 shows in vitro ARF promoter assays. Figure supplement 2 shows additional images for panels B, D, and E. Figure supplement 3 shows wound healing in WT and ARF:GFP fins. Results are shown as mean ± standard deviation. hpa: Hours post amputation.DOI:http://dx.doi.org/10.7554/eLife.07702.003