Fig 2: TRAIL-R2 modulates p53 transcriptional activity independently of caspases.A Whole-cell lysates of A549 wild type (WT) and TRAIL-R2-Sup (TR2 Sup) cells were analyzed by western blotting for the protein levels of TRAIL-R2, p53, MDM2, BAX, and p21. The level of β-Actin was determined in parallel and served as loading control. B mRNA levels of p21, MDM2 and BAX were analyzed by qRT-PCR in A549 cells and normalized to TBP. Bar chart shows mean values ± SD of three biological replicates (n = 3). C Cell cycle analysis through PI staining followed by flow cytometry of A549 WT and TRAIL-R2-Sup cells. Bar chart shows mean values ± SD of three biological replicates (n = 3). D Chromatin Immunoprecipitation (ChIP) was performed with anti-p53 (DO-1) and isotype control antibodies (IgG2a) on chromatin isolated from A549 WT and TRAIL-R2 Sup cells. DNA was extracted, and qRT-PCRs were performed using primers detecting the CDKN1A promotor. Enrichment was calculated as the fold increase in specific signal relative to the background signal. Results are shown ± SEM of four biological replicates (n = 4). E A549 WT and TRAIL-R2 Sup cells were transiently transfected with expression vector coding for the long (TR2-long) or short (TR2-short) isoform of TRAIL-R2, each carrying a point mutation in the death domain, or with an empty vector (pCR3.1). After 48 h, protein levels of TRAIL-R2, p53, and p21 were analyzed by western blotting. The level of β-Actin was determined in parallel and served as loading control. Bands were analyzed by densitometry. Intensity of each band was normalized to the corresponding β-Actin. F A549 cells were treated with zVAD-fmk (20 µM) for 48 h. Whole-cell lysates were analyzed by western blotting for the expression of TRAIL-R2, p53, and p21. The level of β-Actin was determined in parallel and served as loading control. G A549 WT and TRAIL-R2 Sup cells were irradiated with 10 J/m2 of UV-C radiation. After 16 h cell cycle analysis through PI staining followed by flow cytometry was performed. Bar chart shows mean values ± SD of three biological replicates (n = 3). H A549 WT and TRAIL-R2 Sup cells were irradiated with 10 J/m2 of UV-C radiation. After 6 h and 16 h whole-cell lysates were prepared and analyzed by western blotting for the expression of TRAIL-R2, p53, and p21. β-Actin was analyzed in parallel as loading control. *p < 0.05.

Fig 3: TRAIL-R2 affects the p53-mediated transcriptional regulation of the p21 gene (CDKN1A).A HCT116 p53 WT and p53 KO cells were transiently transfected with TRAIL-R2 siRNA (TR2 KD) or control siRNA (Ctrl). After 48 h, protein levels of TRAIL-R2, p53, and p21 were analyzed by western blotting. The level of β-Actin was determined in parallel and served as loading control. Bands were analyzed by densitometry. Intensity of each band was normalized to the corresponding β-Actin. B Relative expression of p21 mRNA levels (normalized to TBP) were analyzed by qRT-PCR in HCT116 p53 WT and p53 KO cells, which were transiently transfected as in (A) for 72 h. Bar chart shows mean values ± SD of three biological replicates (n = 3). C HCT116 p53 WT and p53 KO cells were transfected as in (A) and cell cycle analyses were performed 72 h later. Bar chart shows mean values ± SD of three biological replicates (n = 3). D Schematic representation of reporter vectors used in (E) and (F). E, F HCT116 p53 WT (E, F) and HCT116 p53 KO cells (F) were transfected with siRNA as in (A). After 48 h, cells were additionally transfected in duplicates with plasmids containing a luciferase gene (luc) under control of the p21-promotor (with or without p53-responsive element (RE1 or 2). After 24 h luciferase activity was measured and normalized to the activity of renilla luciferase. Bar chart shows mean values ± SD of one representative experiment (n = 1). ns, non significant; *p < 0.05.

Fig 4: TRAIL-R2 interacts with p53 in the nucleus.Intracellular distribution of TRAIL-R2 and p53 in HCT116 cells was analyzed by indirect immunofluorescence followed by (A) confocal LSM and (B) ImageStream high-throughput microscopy. Scale bar 20 µm. The respective antibody controls are shown in Supplementary Fig. 1. C TRAIL-R1 and TRAIL-R2 were precipitated from nuclear fractions of HCT116 p53 WT and p53 KO cells by receptor-specific antibodies (Mapa—Mapatumumab, anti-TRAIL-R1 antibody, lane 1 and 3; Lexa—Lexatumumab, anti-TRAIL-R2 antibody, lane 2 and 4). As controls, antibodies alone were analyzed in parallel (lane 5, 6). Nuclear lysates and precipitated protein complexes were examined by western blotting (anti-p53 antibody DO-1). As gel loading control the levels of nuclear protein hnRNPA1 was analyzed in parallel. D AsPC-1 p53 null cells were stable transfected with a temperature-sensitive p53-mutant. These cells express mutant-p53 (p53 MT) at 37 °C and wild-type p53 (p53 WT) at 32 °C. Whole-cell lysates of AsPC-1 cells cultured for 24 h at 37 °C or 32 °C were analyzed for the presence of TRAIL-R2, p53 (anti-p53 antibody DO-1) and p21 by western blotting. The level of β-Actin was determined in parallel and served as loading control. E TRAIL-R1 and TRAIL-R2 were precipitated from nuclear fractions of AsPC-1 cells cultured for 24 h at 37 °C or 32 °C. As controls, antibodies alone were analyzed in parallel. Nuclear lysates and precipitated protein complexes were examined by western blotting (anti-p53 antibody DO-1). hnRNPA1 was analyzed in parallel and served as loading control.

Fig 5: SOX2 expression is required for endometrial cancer cell growth via CDKN1A. (a) Western blot analyses. (b) Time course of cancer cell proliferation. (c) Senescence‐associated β‐galactosidase activity analysis in HEC59 cells transfected with SOX2 siRNA or control siRNA. (d) Cell cycle analysis of EN cells was performed using a FACS Aria II instrument after staining of the cells with propidium iodide. (e) CDKN1A mRNA expression levels in HEC59 cells transfected with siRNA targeting SOX2, as analyzed by quantitative real‐time PCR. (f) Chromatin immunoprecipitation assays were used to detect the SOX2‐CDKN1A interaction in HEC59 cells.