Journal: Oncotarget

Article Title: RECQ1 expression is upregulated in response to DNA damage and in a p53-dependent manner

doi: 10.18632/oncotarget.18237

Figure Lengend Snippet: ( A ) RECQ1 expression is upregulated in response to MMS treatment in HCT116 cells expressing wild-type p53. Cells were either untreated or treated for 4 h with the indicated dose of MMS. Fold-change in gene expression compared to untreated and normalized to GAPDH is shown. Two primer sets (RECQ1 #1, and RECQ1 #2) were used for measuring RECQ1 mRNA. β -actin served as an additional housekeeping control. ( B ) Isogenic HCT116 cells expressing either the wild-type p53 (p53 WT) or knockout for p53 (p53 KO) were exposed to MMS (1 mM) for indicated time period and the fold-change in mRNA expression of RECQ1 and p21 compared to untreated as measured by qPCR is shown. ( C ) Fold-change in mRNA expression of RECQ1 and p21 compared to untreated in isogenic p53WT and p53KO RKO cells is shown. ( D ) Following treatment with MMS (1 mM) for indicated time, expression of RECQ1 and p53 proteins was determined by Western blot analysis of whole-cell extracts. GAPDH was used as a loading control and fold-change in RECQ1 protein expression normalized to GAPDH is indicated. ( E ) U2OS cells, 42 h after transfection with control siRNA or p53 siRNA, were exposed to MMS (1 mM) for indicated time period and the fold-change in mRNA expression of RECQ1 and p21 compared to untreated cells as measured by qPCR is shown. β -actin served as an additional housekeeping control. Knockdown of p53 protein level is shown by Western Blot. For all the qPCR data, values are average of three independent experiments and standard deviation is indicated by error bars. Statistical significance of RECQ1 expression changes in untreated versus treatment groups is indicated as * p < 0.05; # p < 0.01; ** p < 0.005; ## p < 0.001; or n. s., non-significant.

Article Snippet: Isogenic pair of p53-wild type (p53WT) and p53-knockout (p53KO) human colon carcinoma cell lines HCT116 and RKO were provided by Dr. B. Vogelstein (The Johns Hopkins Kimmel Cancer Center, Baltimore, MD); human cervical adenocarcinoma HeLa, osteosarcoma U2OS and breast adenocarcinoma MCF7 and MDA-MB-231 cell lines used in this study were purchased from American Type cell culture (ATCC); and mouse embryonic fibroblasts (MEFs) have been described [ ].

Techniques: Expressing, Gene Expression, Control, Knock-Out, Western Blot, Transfection, Knockdown, Standard Deviation

Journal: Oncotarget

Article Title: RECQ1 expression is upregulated in response to DNA damage and in a p53-dependent manner

doi: 10.18632/oncotarget.18237

Figure Lengend Snippet: ( A ) Western blot showing siRNA knockdown of RECQ1 in p53-proficient and p53-deficient HCT116 cells. ( B ) RECQ1-depletion and p53 loss have synergistic effect on survival. Following 48 h of siRNA transfection, p53WT and p53KO-HCT116 cells were exposed to MMS for 24 h and subsequently grown for 24 h in drug-free medium. Surviving fraction compared to untreated was determined by cell count. Knockdown of RECQ1 was confirmed by Western blotting as shown. ( C ) U2OS cells stably transduced with a control (shCTL) or RECQ1 (shRECQ1)-specific shRNA were exposed to MMS for 24 h and subsequently grown for 24 h in drug-free medium. Surviving fraction compared to untreated was determined by cell count. ( D ) Western blot analysis of whole cell extracts of stable control and RECQ1 knockdown U2OS cells, untreated or treated with doxorubicin (1 μM) or MMS (1 mM) for 4 h. A short exposure of RECQ1 Western blot is also included. ( E ) DNA double strand breaks in control or RECQ1-depleted cells. Neutral Comet Assay was used to determine tail moment as a measure of double strand breaks in stable control and RECQ1 knockdown U2OS cells, untreated or treated with doxorubicin (1 μM) or MMS (1 mM) for 4 h. Mean tail moment of untreated shCTL was used to normalize the data and is shown as 100%. Statistical significance of difference in tail moment as compared to untreated shCTL is indicated as * p < 0.05; # p < 0.01; ** p < 0.005 or n. s., non-significant.

Article Snippet: Isogenic pair of p53-wild type (p53WT) and p53-knockout (p53KO) human colon carcinoma cell lines HCT116 and RKO were provided by Dr. B. Vogelstein (The Johns Hopkins Kimmel Cancer Center, Baltimore, MD); human cervical adenocarcinoma HeLa, osteosarcoma U2OS and breast adenocarcinoma MCF7 and MDA-MB-231 cell lines used in this study were purchased from American Type cell culture (ATCC); and mouse embryonic fibroblasts (MEFs) have been described [ ].

Techniques: Western Blot, Knockdown, Transfection, Cell Counting, Stable Transfection, Transduction, Control, shRNA, Neutral Comet Assay

Journal: Oncotarget

Article Title: RECQ1 expression is upregulated in response to DNA damage and in a p53-dependent manner

doi: 10.18632/oncotarget.18237

Figure Lengend Snippet: ( A ) Partial sequence of the RECQ1 promoter (−819 bp to +21 bp) is shown with potential p53-binding sites predicted by Promo 3.0.2 indicated in red, those predicted by Tp53 database are underlined, and those predicted by both Promo 3.0.2 and Tp53 are indicated in bold. Transcriptional start site is indicated as +1. The position of primer used for PCR-cloning of a 625 bp fragment from RECQ1 promoter in pGL3-Basic for luciferase assay is indicated by blue arrow. Three primer sets used for ChIP-qPCR analyses of p53 binding are indicated. ( B ) Dual Luciferase Assay shows p53-dependent transcriptional activation of a 625bp fragment from RECQ1 promoter region; pGL3_p53RE served as a positive control and pGL3_vector as a negative control. The relative luciferase activity was first determined by ratio of firefly and renilla luciferase activity for each sample in p53-WT and p53-KO HCT116 cells, and the relative promoter activity in p53-KO was calculated using the relative luciferase activity from p53-WT cells transfected by each pGL3-basic construct as a reference of 1. Bars indicate mean values plus standard deviation of three independent experiments. ( C ) MMS-induced enrichment of p53 to RECQ1 promoter. HCT116 cells, p53-WT or p53-KO, untreated or treated with MMS (1 mM, 8 h), were processed for ChIP using a p53-specific antibody. ChIP experiments with rabbit IgG served as negative control. ChIP-qPCR of immunoprecipitated DNA with three probes specific for RECQ1 promoter sequence containing predicted p53 binding sites (#1, #2, and #3, and as shown in A). Binding of p53 to p21 promoter containing p53RE served as a positive control. Fold enrichment over IgG was determined and is shown for each primer pair for the ChIP. Relative occupancy at RECQ1 and p21 promoter sequence versus a negative control site DNA containing GAPDH shows MMS treatment induced enrichment of p53. Statistical significance of enrichment in untreated versus treatment groups is indicated. Results are expressed as means ± SEM for at least three independent experiments. ( D ) A representative agarose gel of the amplified DNA immunoprecipitated with p53 antibody shows MMS-induced enrichment of p53 to RECQ1 promoter whereas MMS treatment did not change GAPDH abundance in p53 ChIP. M, DNA size marker. ( E ) Enhanced recruitment of RNA POL II to RECQ1 promoter following MMS treatment. HCT116 cells, p53-WT or p53-KO, untreated or treated with MMS (1 mM, 8 h), were processed for ChIP using a RNA POL II-specific antibody or rabbit IgG. Binding of RNA POL II to RECQ1 promoter sequence and p21 promoter was measured by qPCR of immunoprecipitated DNA. Fold enrichment over IgG was determined and is shown for each primer pair for the ChIP. Relative occupancy at RECQ1 and p21 promoter sequence versus a negative control non-promoter GAPDH sequence shows MMS treatment induced enrichment of RNA POL II. Statistical significance of enrichment in untreated versus treatment groups is indicated. Results are expressed as means ± SEM for at least three independent experiments.

Article Snippet: Isogenic pair of p53-wild type (p53WT) and p53-knockout (p53KO) human colon carcinoma cell lines HCT116 and RKO were provided by Dr. B. Vogelstein (The Johns Hopkins Kimmel Cancer Center, Baltimore, MD); human cervical adenocarcinoma HeLa, osteosarcoma U2OS and breast adenocarcinoma MCF7 and MDA-MB-231 cell lines used in this study were purchased from American Type cell culture (ATCC); and mouse embryonic fibroblasts (MEFs) have been described [ ].

Techniques: Sequencing, Binding Assay, PCR Cloning, Luciferase, ChIP-qPCR, Activation Assay, Positive Control, Plasmid Preparation, Negative Control, Activity Assay, Transfection, Construct, Standard Deviation, Immunoprecipitation, Agarose Gel Electrophoresis, Amplification, Marker

Journal: Oncotarget

Article Title: RECQ1 expression is upregulated in response to DNA damage and in a p53-dependent manner

doi: 10.18632/oncotarget.18237

Figure Lengend Snippet: ( A , B ) RECQ1 expression is upregulated in response to Temozolomide (TMZ) or Fotemustine (FMS) treatment of HCT116 cells expressing wild-type p53. RECQ1 mRNA expression was measured using two independent primer sets; fold-change in expression compared to untreated and normalized to GAPDH is shown. β -actin served as an additional housekeeping control. Values are average of three independent experiments and standard deviation is indicated by error bars. Statistical significance of RECQ1 expression changes in untreated versus treatment groups is indicated as * p < 0.05; # p < 0.01; ** p < 0.005; ## p < 0.001; or n. s., non-significant. ( C ) Following 48 h of control or RECQ1 siRNA transfection, p53WT and p53KO-HCT116 cells were exposed to increasing dose of TMZ for 24 h and subsequently grown for 24 h in drug-free medium. Surviving fraction compared to untreated was determined by cell count. Knockdown of RECQ1 was confirmed by Western blotting (not shown). Surviving fraction values are the mean ± SEM from three independent experiments. ( D ) RECQ1 expression is also upregulated in U2OS cells following treatment with TMZ (1 mM, 6 h) or FMS (32 μM, 6 h). Fold-change in expression compared to untreated and normalized to GAPDH is shown. Values are average of three independent experiments and standard deviation is indicated by error bars. Statistical significance of change in RECQ1 expression compared to untreated is indicated. ( E , F ) Stable control (shCTL) and RECQ1 knockdown (shRECQ1) U2OS cells were exposed to increasing dose of TMZ or FMS for 24 h and subsequently grown for a further 24 h in drug-free medium. The graphs show the cellular surviving fractions measured at different doses of drug treatment in control and RECQ1-depleted cells. Surviving fraction values are the mean ± SEM from three independent experiments. ( G ) Whole cell extracts prepared from stable control and RECQ1 knockdown U2OS and MCF7 cells cultured for 5 days in the absence or presence of TMZ (100 μM) were subjected to Western blot analysis of cleaved PARP for apoptosis. Knockdown of RECQ1 was verified by Western blotting. TMZ treatment lead to increase in RECQ1 protein level in both U2OS and MCF7 cells transduced with control shRNA.

Article Snippet: Isogenic pair of p53-wild type (p53WT) and p53-knockout (p53KO) human colon carcinoma cell lines HCT116 and RKO were provided by Dr. B. Vogelstein (The Johns Hopkins Kimmel Cancer Center, Baltimore, MD); human cervical adenocarcinoma HeLa, osteosarcoma U2OS and breast adenocarcinoma MCF7 and MDA-MB-231 cell lines used in this study were purchased from American Type cell culture (ATCC); and mouse embryonic fibroblasts (MEFs) have been described [ ].

Techniques: Expressing, Control, Standard Deviation, Transfection, Cell Counting, Knockdown, Western Blot, Cell Culture, Transduction, shRNA

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