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: Cell Division

Article Title: FAT 10, a gene up-regulated in various cancers, is cell-cycle regulated

doi: 10.1186/1747-1028-1-20

Figure Lengend Snippet: FAT10 expression at the transcript level and promoter is cell-cycle regulation in HCT116 cells . (A) Cell-cycle profiles of HCT116 cells synchronized at the various cell-cycle stages: C – Asynchronous cells; G1-Cells synchronized to G1-phase with L-minosine; S-Cells syncrhonized to S-phase with Thymidine and G2/M-Cells synchronized to G2/M phase with nocodazole. (B) FAT10 protein expression normalized against β-actin protein levels. Top panel: Western blot of a representative experiment. Bottom panel: mean and standard deviation of results quantitated from Western blots from three independent experiments. *p < 0.05. (C) FAT10 mRNA transcript levels normalized against β-actin mRNA levels. Results shown are the mean and standard deviation from three independent experiments. *p < 0.05 and **p < 0.01. (D) Promoter-reporter construct used to assay FAT10 promoter activity. Various lengths of the FAT10 promoter drive the β-galactosidase reporter gene while the constitutive CMV promoter drives the EGFP to normalize for differences in transfection efficiencies. (E) Left panel – FAT10 promoter activity in HCT116 cells expressed as normalized β-galactosidase activity from 3 independent experiments. Middle Panel – Pictorial representation of the various regions of DNA upstream and downstream the transcription start site of the FAT10 gene that is cloned before the β-galactosidase gene. Right Panel – FAT10 promoter activity in HCT116 cells synchronized to the various cell-cycle stages (white: G2/M; black: S and Grey: G1) in HCT116 cells. Results shown are the mean and standard deviation from three independent experiments.

Article Snippet: Western blot analyses was performed on HCT116 cell lysates (7 μg/well) (Lee et al., 2003) using the rabbit anti-FAT10 (Lee et al., 2003), and goat anti-actin (Santa Cruz Biotechnology) primary antibodies and HRP-conjugated anti-mouse or anti-goat IgG secondary antibodies (Pierce Biotechnology).

Techniques: Expressing, Western Blot, Standard Deviation, Construct, Activity Assay, Transfection, Clone Assay