Fig 2: AHNAK mediates the EMT via TP53 signaling pathway. A: Western blot analysis showed that after knockdown of AHNAK, only expression of TP53 was altered, while other markers, including AKT and ERK. The phosphorylated level of TP53, AKT and ERK also showed no alterations. B: qRT-PCR showed the alteration of markers in TP53 signaling pathway. C: GSEA suggested an enrichment of TP53 signaling pathway in PDAC by analyzing the GEO database. D: Western blot showed after the recombinant TP53 protein was added into the AHNAK deficiency cell lines, E-cad, the epithelial markers of EMT phenotype was restored. E and F: CCK-8 and Transwell assays showed that recombinant TP53 could restore cellular proliferation and migration.

Fig 3: A diagram illustrates the signaling mechanisms of how SAHA may be inhibiting GSCs. Low-dose of SAHA increases both p53 protein and its phosphorylation at Ser33 via p38 activation, leading to a rise in cell cycle checkpoint activation and cellular sensecence, but high-dose of SAHA induces p53 Ser15 phosphorylation and caspase-mediated involving both the caspase-8 and the caspase-9 pathways in GSCs

Fig 4: Interaction of TRIP13 with TTC5 regulates p53 signaling and apoptosis in renal cells.(A) Representative co-immunoprecipitation using myc antibody with protein lysates from 293T cells containing over-expression of myc-TTC5 and EGFP-TRIP13. Membranes were probed for TTC5 (top panel) and EGFP (bottom panel). Input = 10% of the total lysate. EGFP = EGFP-expressing plasmid as a control. (B) Immunocytochemistry of IMCD-3 cells transfected with a plasmid encoding Trip13 cDNA fused to EGFP. Green fluorescence indicates the predominant cytoplasmic localization of TRIP13. TTC5 immunostaining was detected by TTC5 specific antibody with AlexaFluor488 (red). The images were merged with DAPI (blue). Scale bar = 25 µm.(C) Immunofluorescence in IMCD-3 cells was performed with EGFP-TRIP13 in the absence (top panels) or presence (bottom panels) of TTC5. DAPI stain to indicate nuclei. Images are merged to demonstrate re-distribution of some cytoplasmic TRIP13 to the nucleus. (D) Subcellular fractionation of 293T cells over expressing either TRIP13, TTC5 or both and detection by immunoblot analysis. A representative immunoblot analysis was performed to measure the total p53 levels in each enriched fraction. As a negative control, EGFP-expressed cell lysates were separated for each subcellular fraction. C = cytoplasm; M = membrane; and N = nuclear fraction. Graphical analysis of the band intensities as a ratio of cytoplasmic:nuclear fraction is shown. (E) Localization of TRIP13 in the tubular epithelial cells. EGFP-TRIP13 plasmid was transfected into IMCD-3 cells, and a mixed population of stable EGFP-TRIP13 expressing cells was generated following selection with Geneticin (500-750 µg/mL). The effect of a sub-lethal dose of H2O2 (8.8 µM) in the presence and absence of importazole (IMPZ) (50–75 µM) was analyzed. DMSO was used as a vehicle control. (F) Proposed schematic by which TRIP13 regulate p53 signaling and apoptosis of renal epithelial cells following biological injury through a DNA damage-mediated pathway.

Fig 5: SAHA induces GSCs senescence via activation of the p38-p53 pathway. a to c GSCs from U87MG cells were collected 7 days after treatment of SAHA at different doses. The cellular lysates were then analyzed by Western blotting with indicated antibodies (a). The senescence-associated ß-galactosidase (SA-ß-gal) activities were then examined by SA-ß-gal kit (Cell Signaling, details described in materials and methods). Photomicrographs of spheroid bodies were randomly selected in microscopic fields (b) and the percentage of SA-ß-gal positive cells was quantified; bar, S.D.; ** p < 0.01 (c). d to g GSCs were pre-treated with or without 3 µM SB203580 (SB) for 1 h and then treated with DMSO or with SAHA (1 µM in G, 2.5 µM in d to g) for 7 days. After treatment, cellular senescence was measured by the detection of SA-ß-gal activity (d). The percentage of SA-ß-gal positive cells was quantified; bar, S.D., *, P < 0.05, **, P < 0.01 (e). The protein lysates were analyzed by Western blotting with indicated antibodies (f and g). h GSCSs were treated with DMSO or with different doses of SAHA as indicated for 2 days. After treatment, the protein lysates were analyzed by Western blotting