Fig 2: Cas9-CRISPR effectively targeted the CDKN2A locus. (a) Schematic representation of the CDKN2A locus and primer sets flanking the sgRNA target sites. (b) PCR amplification of the CDKN2A locus with primer sets flanking predicted Cas9 cut sites. Genomic DNA obtained from each clone and PrEC was amplified with the primer sets indicated above and resolved by agarose electrophoresis. A DNA ladder is shown in the first lane of the Exon 2 gel. All products match their predicted sizes. Gel Images are cropped for clarity. Images corresponding to full-length gels are shown in Suppl. Figure 1B. (c) hTERT is overexpressed in all the cell lines. hTERT mRNA expression was determined by RNA-Seq as described in the text and Fig. 6. Represented is the variance stabilization transformed normalized average (VST-averages) for hTERT in PrEC compared to all the immortalized cell lines.

Fig 3: Expression level of KIF11 and CDKN2A (p16 and p14) in HCC. (A) Based on the mRNA expression profile data and clinical information in the TCGA database, KIF11 expression in HCC tissues and associated adjacent tissues were displayed in boxplots. Protein (B) and mRNA (C) level of KIF11, p16 and p14 in cultured normal liver cells (HLSEC and THLE‐3) and hepatoma cells (HCCLM3, Huh7, HepG2, SNU398). (D) Correlation analysis of KIF11 and p16 in cultured normal liver cells (HLSEC and THLE‐3) and hepatoma cells (HCCLM3, Huh7, HepG2, SNU398). (E) Correlation analysis of KIF11 and p14 in cultured normal liver cells (HLSEC and THLE‐3) and hepatoma cells (HCCLM3, Huh7, HepG2, SNU398). (F) Correlation analysis of KIF11 and p16 in ROS stress‐induced normal liver cells (THLE‐3) and hepatoma cells (Huh7 and HepG2). (G) Correlation analysis of KIF11 and p14 in ROS stress‐induced normal liver cells (THLE‐3) and hepatoma cells (Huh7 and HepG2). (H) Pie chart of different expression levels of KIF11, p16 and p14 in clinical HCC tissues. A total of 83 clinical cases were analysed. 2 cases were KIF11high p16low, 15 cases were KIF11high p14low, 33 cases were KIF11high p16low p14low, 13 cases were KIF11low p16high, 3 cases were KIF11low p14high, 8 cases were KIF11low p16high p14high and 9 cases were KIF11high p16high p14high /KIF11low p16low p14low. (I) IHC pictures of KIF11high p16low expression in patient 1, IHC pictures of KIF11high p14low expression in patient 2 and KIF11high p16low p14low expression in patient 3. The scale bar indicates 50 μm. Data shown are the mean ± SD (n ≥ 3; *p < .05, **p < .01, ***p < .001, two‐tailed t‐test).

Fig 4: Comparative analyses of PrEC immortalized Clones 1, 2, 3 and p16-KO growing in 3D Cultures. (a) Representative images of each immortalized hPrEC clone and ATCC hPrEC P5 seeded in between two layers of Matrigel in iBidi μ-slide for 9 days (clones) or 12 days (PrEC). Organoid images taken using EVOS inverted microscope, at indicated fields. Note the formation of lumens in each clone. (b) Representative images of each PrEC immortalized clone and ATCC hPrEC P5 seeded between two layers of Matrigel and stained with laminin, vimetin (EMT marker), CK5 (Basal marker), Integrin B, E-cadherin (Epithelial Marker), CK18 (Luminal Marker). All organoids were also stained with phalloidin (F-actin) and DAPI. Images taken at 63 × objective (Zoom factor .75) using Leica TCS SP8 Confocal Microscope.

Fig 5: STC p15INK4Bexpression in vitro. (A) SA-β-Gal staining in two cancer cell senescence models, H2O2-induced (60 μM) and doxorubicin (Dox)-induced (50 nM) senescence models (n = 4, each). The p values are obtained using Mann-Whitney U test. (B) DAPI staining revealed heterochromatin foci, in both H2O2-induced (60 μM) and doxorubicin (Dox)-induced (50 nM) senescence models. The proportion of SAHF-positive cells out of total cells was shown in the bar graph and p values were obtained from chi-square test. (C) mRNA expression of CDKN2A and CDKN2B in H2O2-induced (60 μM) and doxorubicin (Dox)-induced (50 nM) senescence models (n = 3, each). The p values were obtained using Student's t-test.