Journal: Oncogene

Article Title: Transglutaminase 2 promotes tumorigenicity of colon cancer cells by inactivation of the tumor suppressor p53

doi: 10.1038/s41388-021-01847-w

Figure Lengend Snippet: A – C Gene expression profiling by RNA-seq of SW480 cells after transduction with either shTGM2-1 or shSCRMBL. A Unsupervised hierarchical clustering of the top 1000 differentially expressed genes (DEGs) upon TGM2 knockdown across the four biological replicates. B MA plot relating p values for all differentially expressed genes between shTGM2-1 and shSCRMBL from four biological replicates. Red dots indicate significantly regulated genes (adjusted P < 0.05). List of regulated genes is presented in Supplementary Table S . C Scatter plot of gene set enrichment analysis of DEGs relating the Q-value for Hallmark gene-set signatures. The top 16 enriched pathways are shown ( P < 0.05, Fold change ≥2). The color and size of each dot represent the Rich factor and the number of DEGs mapped to the indicated pathway, respectively. D Proteome analysis of regulated proteins involved in apoptosis upon shRNA-mediated TGM2 knockdown. Representative blot of Proteome Profiler Array™-Human Apoptosis Array analysis of SW480 cells. The regulation of protein expression of phosphorylated p53 variants is shown. E – H Quantification of p53 and phosphorylated p53 (S15, S46, and S392) upon TGM2 knockdown in SW480 ( E , G ) and HCT-116 ( F , H ) cells via Simple Western technology ( n = 3; Mann–Whitney U test).

Article Snippet: The p53 knockout colon cancer cell line HCT-116 (p53 −/− ) was obtained from Accegen Biotechnology (Köln, Germany).

Techniques: Gene Expression, RNA Sequencing, Transduction, Knockdown, shRNA, Expressing, Simple Western, MANN-WHITNEY

Journal: Oncogene

Article Title: Transglutaminase 2 promotes tumorigenicity of colon cancer cells by inactivation of the tumor suppressor p53

doi: 10.1038/s41388-021-01847-w

Figure Lengend Snippet: A Representative images of proximity ligation assay (PLA) of TGM2 and p53 in SW480 cells. Cells incubated only with TGM2 antibody served as negative control (I). Protein–protein interaction of TGM2 and p53(S15) was visualized using hybridization probes labeled with Texas Red (II). Nuclei were stained with DAPI (blue). B Quantification of TGM2-p53 interaction and associated technical controls (Ctrl). Technical controls demonstrate the specificity of PLA signals. Each dot represents one cell. Mean value of PLA dots per cell is shown by the black line. C Representative images of proximity ligation assay of TGM2 and p53 in patient-derived normal epithelial cells (I) and corresponding colon cancer cells (II). D Quantification of TGM2-p53 interaction in primary patient material. (Significance was calculated using Kruskal–Wallis test). E Co-immunoprecipitation (Co-IP) of endogenous TGM2 and p53 or phosphorylated p53(S15) in SW480, HCT-116 p53 wildtype cells (wt) and HCT-116 p53 knockout cells (−/−). F Super-resolved image of a HCT-116 cell immunostained for TGM2 (red) and p53(S15) (cyan). A zoom-in of the highlighted region is shown on the right. White regions indicate overlapping signal of TGM2 and p53(S15) (yellow arrowheads). Scale bars represent 5 µm and 1 µm, respectively.

Article Snippet: The p53 knockout colon cancer cell line HCT-116 (p53 −/− ) was obtained from Accegen Biotechnology (Köln, Germany).

Techniques: Proximity Ligation Assay, Incubation, Negative Control, Hybridization, Labeling, Staining, Derivative Assay, Immunoprecipitation, Co-Immunoprecipitation Assay, Knock-Out

Journal: Oncogene

Article Title: Transglutaminase 2 promotes tumorigenicity of colon cancer cells by inactivation of the tumor suppressor p53

doi: 10.1038/s41388-021-01847-w

Figure Lengend Snippet: A – C HCT-116 p53 wildtype cells (wt) and HCT-116 p53 knockout cells (−/−) were transduced with either shTGM2-1, shTGM2-2, or shSCRMBL. Time-lapse imaging and proliferation assay were performed to determine a rescue from cell death upon TGM2 knockdown. A Fold change of cell number of HCT-116 p53 wt and HCT-116 p53 −/− cells upon TGM2 knockdown in comparison to shSCRMBL control determined at day three after transduction. Data are presented as mean ± SD of three independent experiments (** P < 0.01, Mann–Whitney U test). B Single cell tracking of HCT-116 p53 wt and HCT-116 p53 − /− cells after TGM2 knockdown with shTGM2-1 and (C) shTGM2-2. Cumulative cell death events are shown over time (*** P < 0.001, Log-rank test). D Direct visualization of p53 activation upon TGM2 knockdown by time-lapse video-microscopy. Sequence of phase contrast images, tdTOMATO fluorescence of shTGM2-1 and p53-driven destabilized GFP reporter , depicting the same field of view over the time course of 30 hours as indicated in the corresponding panels in I–VIII. The yellow circles designate tracked cells over time. (I–VIII) show corresponding sequence of fluorescence images taken at the same time points as the phase contrast images. (I) Shown are two representative HCT-116 cells. (II and III) 6-8 hours after lentiviral transduction of shTGM2-1 both HCT-116 cells express the red fluorescent tdTOMATO reporter, indicating a knockdown of TGM2. (IV-VI) Another 4–10 hours later both cells express the green fluorescent (GFP) p53 reporter, indicating the induction of p53 activity. (VII and VIII) About 24 hours after transduction both HCT-116 cells subsequently undergo apoptosis (white arrows). Movie S shows all assembled images (3 min temporal resolution) of the same sequence.

Article Snippet: The p53 knockout colon cancer cell line HCT-116 (p53 −/− ) was obtained from Accegen Biotechnology (Köln, Germany).

Techniques: Knock-Out, Transduction, Imaging, Proliferation Assay, Knockdown, Comparison, Control, MANN-WHITNEY, Single Cell Tracking, Activation Assay, Microscopy, Sequencing, Fluorescence, Activity Assay

Journal: Journal of Biological Inorganic Chemistry

Article Title: Revisiting the anticancer properties of phosphane(9-ribosylpurine-6-thiolato)gold(I) complexes and their 9H-purine precursors

doi: 10.1007/s00775-022-01968-x

Figure Lengend Snippet: Inhibitory concentrations IC 50 [µM] of 6-MP, 6-TG, 6-MMR, auranofin and complexes 1 – 9 when applied to EA.hy926 endothelial hybrid cells and cells of human HCT-116, HCT-116 p53−/− (p53 knockout mutant) and HT-29 colon carcinomas, HeLa and mdr KB-V1 Vbl cervix carcinoma (treated with and without 1 µM verapamil), MCF-7 and mdr MCF-7 Topo mamma carcinoma, U-87 glioblastoma, 518A2 melanoma, and human adult dermal fibroblasts HDFa

Article Snippet: 518A2 human melanoma cells (Department of Radiotherapy & Radiobiology, University Hospital Vienna, Austria), HCT116 wt (DSMZ ACC-581) and its HCT116 p53−/− knockout mutant colon carcinoma cells, U87 glioblastoma cells (ATCC HTB-14), EA.hy926 somatic cell hybrid cells (ATCC CRL-2922), HeLa cervix carcinoma cells (DSMZ ACC-57), MCF-7 breast cancer cells (DSMZ ACC-115), HT-29 cisplatin resistant colon cancer cells (DSMZ ACC-299) and non-malignant human dermal fibroblasts HDFa (ATCC PCS-201-012) were cultured in Dulbecco’s modified Eagle medium (PAN biotech), supplemented with 10% (v/v) fetal bovine serum (Sigma-Aldrich) and 1% (v/v) ZellShield (Minerva Biolabs) at 37 °C under 95% humidity and 5% CO 2 .

Techniques: Knock-Out, Mutagenesis

Journal: Acta Biochimica et Biophysica Sinica

Article Title: R-loop formation contributes to mTORC1 activation-dependent DNA replication stress induced by p53 deficiency

doi: 10.3724/abbs.2024188

Figure Lengend Snippet: Establishment of p53 -knockout cell line by CRISPR-Cas 9 (A) Guide-RNA targeted location is p53 gene Exon 4 (86–105), the specificity and efficiency score of gRNA sequence were evaluated via the Benchling web site. (B) p53 knockout was done in mNS-5 cell line and HCT 116 cell line. p53 protein level was confirmed by western blot analysis. (C) p53 knockout efficiency was confirmed in HCT116 cells by immunostaining using P53(DO-7) mouse mAb (CST #48818) and Alex 488 (green) for p53 labeling, and DAPI staining for nucleus (blue). Scale bar: 25 μm.

Article Snippet: Establishment of p53 -knockout cell line by CRISPR-Cas 9 (A) Guide-RNA targeted location is p53 gene Exon 4 (86–105), the specificity and efficiency score of gRNA sequence were evaluated via the Benchling web site. (B) p53 knockout was done in mNS-5 cell line and HCT 116 cell line. p53 protein level was confirmed by western blot analysis. (C) p53 knockout efficiency was confirmed in HCT116 cells by immunostaining using P53(DO-7) mouse mAb (CST #48818) and Alex 488 (green) for p53 labeling, and DAPI staining for nucleus (blue).

Techniques: Knock-Out, CRISPR, Sequencing, Western Blot, Immunostaining, Labeling, Staining