Journal: Molecular Medicine Reports

Article Title: Tissue factor signalling modifies the expression and regulation of G1/S checkpoint regulators: Implications during injury and prolonged inflammation

doi: 10.3892/mmr.2024.13404

Figure Lengend Snippet: HDBECs (2×10 5 ) were incubated for 24 h with recombinant TF (0, 0.5 and 2 U/ml) or PAR2-AP (SLIGKV; 20 µM), or with recombinant TF (0.5 U/ml) that was pre-incubated for 60 min with 10H10 or HTF1 antibodies (20 µg/ml). Groups of cells were also pre-incubated for 60 min with AIIB2 or SAM11 antibodies (20 µg/ml), prior to the addition of TF. The cells were harvested after 24 h, into separate aliquots (1×10 5 cells). Total RNA was isolated from one group and the mRNA quantified by RT-qPCR, against β-actin. Other aliquots were analysed by western blotting. The data show the amounts of (A) CDK interacting protein/Wildtype p53-activated fragment p21 CIP1/WAF1 mRNA (n=5), and (B) the relative amounts of p21 CIP1/WAF1 protein (n=3) calculated from (C) the western blots of p21 CIP1/WAF1 protein, (using a mouse anti-human antibody) and against GAPDH. TF, tissue factor; PAR2, protease-activated receptor 2.

Article Snippet: The membranes were probed overnight at 4°C with either a goat anti-human p16 antibody (1:2,000 v/v; cat. no. AF5779; R&D Systems Europe, Ltd.), a mouse anti-human p21 antibody (WA-1; cat. no. MCA2325; Bio-Rad Laboratories, Inc.), a rabbit anti-human p14 antibody (cat. no. abx013162; Abbexa, Ltd.) or a rabbit anti-human Cyclin E1 antibody (cat. no. abx012757; Abbexa, Ltd.), each diluted 1:3,000 (v/v) in TBST.

Techniques: Incubation, Recombinant, Isolation, Quantitative RT-PCR, Western Blot

Journal: Molecular Medicine Reports

Article Title: Tissue factor signalling modifies the expression and regulation of G1/S checkpoint regulators: Implications during injury and prolonged inflammation

doi: 10.3892/mmr.2024.13404

Figure Lengend Snippet: Immortalised human epithelial cells (human telomerase reverse transcriptase-human pancreatic nestin-expressing ductal cells) were cultured in 25 cm 2 flasks and supplemented with recombinant TF (0.5 U/ml) or were untreated. The cells were harvested on the indicated wks, total RNA was isolated from one group and the mRNA quantified by RT-qPCR, against β-actin. Other aliquots were analysed by western blotting. The data show the amounts of (A) CDK interacting protein/Wildtype p53-activated fragment p21 CIP1/WAF1 mRNA (n=3), and (B) the relative amounts of p21 CIP1/WAF1 protein (n=3) calculated from (C) the western blots of p21 CIP1/WAF1 protein (using a mouse anti-human antibody) and against GAPDH. TF, tissue factor; Wk, week.

Article Snippet: The membranes were probed overnight at 4°C with either a goat anti-human p16 antibody (1:2,000 v/v; cat. no. AF5779; R&D Systems Europe, Ltd.), a mouse anti-human p21 antibody (WA-1; cat. no. MCA2325; Bio-Rad Laboratories, Inc.), a rabbit anti-human p14 antibody (cat. no. abx013162; Abbexa, Ltd.) or a rabbit anti-human Cyclin E1 antibody (cat. no. abx012757; Abbexa, Ltd.), each diluted 1:3,000 (v/v) in TBST.

Techniques: Reverse Transcription, Expressing, Cell Culture, Recombinant, Isolation, Quantitative RT-PCR, Western Blot

Journal: Molecular Medicine Reports

Article Title: Tissue factor signalling modifies the expression and regulation of G1/S checkpoint regulators: Implications during injury and prolonged inflammation

doi: 10.3892/mmr.2024.13404

Figure Lengend Snippet: Proposed model for the mechanism by which the level of TF on the cell surface may have differential outcomes on G1/S checkpoint regulation. The presence of TF on the cell surface differentially upregulates the expression of Inhibitor of CDK p16 INKa , CDK interacting protein/Wildtype p53-activated fragment p21 CIP1/WAF1 and Alternative reading frame p14 ARF , which is dependent on the concentration of TF and the ability of the cell to dissipate excess TF. Therefore, alterations in p21 CIP1/WAF1 are highly effective in the regulation of the cellular response to acute stress. The interplay between these proteins modulates the signal permitting passage through the cell cycle, or alternatively its arrest. Consequently, the concentration of TF may be an ideal gauge for determining the level of cellular damage. However, the adaptive loss of p16 INK4a function may be promoted by prolonged inflammation leading to permissive transition through the G1/S checkpoint, even in the absence of mutational loss of p16 INKa . TF, tissue factor; PAR2, protease-activated receptor 2, Rb, retinoblastoma protein; E2F, early region 2 binding factor; p21 CIP1/WAF1 , CDK interacting protein/wildtype p53-activated fragment; p16 INK4a , inhibitor of CDK; p14 ARF , alternative reading frame.

Article Snippet: The membranes were probed overnight at 4°C with either a goat anti-human p16 antibody (1:2,000 v/v; cat. no. AF5779; R&D Systems Europe, Ltd.), a mouse anti-human p21 antibody (WA-1; cat. no. MCA2325; Bio-Rad Laboratories, Inc.), a rabbit anti-human p14 antibody (cat. no. abx013162; Abbexa, Ltd.) or a rabbit anti-human Cyclin E1 antibody (cat. no. abx012757; Abbexa, Ltd.), each diluted 1:3,000 (v/v) in TBST.

Techniques: Expressing, Concentration Assay, Binding Assay

Journal: Scientific Reports

Article Title: Polymorphisms in the 5′ upstream regulatory region of p21 WAF1/CIP1 and susceptibility to oesophageal squamous cell carcinoma

doi: 10.1038/srep22564

Figure Lengend Snippet: genotyped in the current study. ( A ) Gene map and polymorphisms in the p21 gene on chromosome 6p21.2. Coding exons are indicated with black blocks and 5′- and 3′-UTRs with white blocks. The first base of the transcription start site is denoted as + 1. ( B ) LD blocks between SNPs in the promoter region of p21 in 96 healthy Han Chinese individuals. Each diamond represents the correlation (D′) between each pair of SNPs. A standard colour scheme is used to display the LD pattern, where dark grey indicates very strong LD; white indicates no LD; and bright grey and shades of grey represent intermediate LD, with an increasing intensity of grey indicating an increasing degree of LD.

Article Snippet: Following 10% goat serum blocking, tissue sections were then incubated with Mouse Anti-Human P21 WAF1 protein, (Biogenex, San Raman, CA) at 4 °C overnight.

Techniques:

Journal: Scientific Reports

Article Title: Polymorphisms in the 5′ upstream regulatory region of p21 WAF1/CIP1 and susceptibility to oesophageal squamous cell carcinoma

doi: 10.1038/srep22564

Figure Lengend Snippet: Haplotype structure of the p21 gene in Chinese population.

Article Snippet: Following 10% goat serum blocking, tissue sections were then incubated with Mouse Anti-Human P21 WAF1 protein, (Biogenex, San Raman, CA) at 4 °C overnight.

Techniques:

Journal: Scientific Reports

Article Title: Polymorphisms in the 5′ upstream regulatory region of p21 WAF1/CIP1 and susceptibility to oesophageal squamous cell carcinoma

doi: 10.1038/srep22564

Figure Lengend Snippet: Risk of ESCC associated with p21 rs3829963 and rs2395655 by smoking status.

Article Snippet: Following 10% goat serum blocking, tissue sections were then incubated with Mouse Anti-Human P21 WAF1 protein, (Biogenex, San Raman, CA) at 4 °C overnight.

Techniques: Control

Journal: Scientific Reports

Article Title: Polymorphisms in the 5′ upstream regulatory region of p21 WAF1/CIP1 and susceptibility to oesophageal squamous cell carcinoma

doi: 10.1038/srep22564

Figure Lengend Snippet: ( A ) No–anti p21, negative control with primary antibody replaced by PBS. ( B ) Anti–p21, the brown signals represent positive staining for P21 protein and the staining was scored on a scale as indicated in the Materials and Methods. Positive cases were defined as those with over 10% of examined cells stained. Magnification, ×400.

Article Snippet: Following 10% goat serum blocking, tissue sections were then incubated with Mouse Anti-Human P21 WAF1 protein, (Biogenex, San Raman, CA) at 4 °C overnight.

Techniques: Negative Control, Staining

Journal: Scientific Reports

Article Title: Polymorphisms in the 5′ upstream regulatory region of p21 WAF1/CIP1 and susceptibility to oesophageal squamous cell carcinoma

doi: 10.1038/srep22564

Figure Lengend Snippet: Association of rs3829963 and rs2365955 with P21 expression of ESCC patients.

Article Snippet: Following 10% goat serum blocking, tissue sections were then incubated with Mouse Anti-Human P21 WAF1 protein, (Biogenex, San Raman, CA) at 4 °C overnight.

Techniques: Expressing

Journal: Cell Death & Disease

Article Title: The pharmalogical reactivation of p53 function improves breast tumor cell lysis by granzyme B and NK cells through induction of autophagy

doi: 10.1038/s41419-019-1950-1

Figure Lengend Snippet: a MDA-MB231 cells express high level of mutated p53 (p53 R280K ) lacking transcriptional activity as shown by the absence of p53-dependent transactivation of its target gene p21 even after γ-irradiation. b , c 24-48 h treatment with CP-31398 reactivates p53 transcriptional activity in MDA-MB231 cells as shown by induction of p21 expression at both mRNA ( b ) and protein ( c ) levels. d – f CP-31398-induced apoptosis and ROS production were evaluated after 48 h treatment using AnnexinV/PI ( d , e ) or DHE ( f ) staining. g , h CP-31398 treatment (48 h) increases MDA-MB231 cell susceptibility to NK-mediated lysis. 51 Cr release assay using NK92 ( d ) or NK cells isolated from a healthy donor ( e ) co-cultured with target cells at different E:T ratios are shown. i CP-31398 treatment increases MDA-MB231 cell susceptibility to NK-mediated lysis in a time dependent manner. A representative 51 Cr release assay (from two independent experiments) using NK cells isolated from a healthy donor is shown. j , k PFT-α (an inhibitor of p53 transcriptional activity) inhibits CP-31398-dependent induction of p21 expression in MDA-MB231 cells at both mRNA ( j ) and protein ( k ) levels. l , m PFT-α inhibits the increase of MDA-MB231 lysis by NK cells observed when CP-31398 is used alone. 51 Cr release assay using NK92 ( l ) or NK cells extracted from a healthy donor ( m ) co-cultured with target cells at the E:T ratio of 50:1 or 30:1, respectively, are shown. n – p The knockdown of mutated p53 expression using siRNA inhibits CP-31398-dependent induction of p21 expression in MDA-MB231 cells at both mRNA ( n ) and protein ( o ) levels and abrogate the CP-31398-dependent increase of MDA-MB231 lysis by NK cells ( p ). Data are representative of at least three independent experiments ( a , c , d , k , o ) or are the mean ± s.d. of three ( e – h , l , m , p ) or five ( b , j , n ) independent experiments. The p values (* P < 0.05; ** P < 0.01) were determined by unpaired two-tailed Student's t test ( b , e , g , h , j , l – n ) or one-way ANOVA ( p )

Article Snippet: From Merck: anti-p21 mouse mAb (clone OP64), anti-mdm2 mouse mAb (clone 2A10).

Techniques: Activity Assay, Irradiation, Expressing, Staining, Lysis, Release Assay, Isolation, Cell Culture, Two Tailed Test

Journal: Cell Death & Disease

Article Title: The pharmalogical reactivation of p53 function improves breast tumor cell lysis by granzyme B and NK cells through induction of autophagy

doi: 10.1038/s41419-019-1950-1

Figure Lengend Snippet: a CP-31398 treatment (48 h) increases p21 and Mdm2 expression by MDA-MB231 cells, does not alter Bax level (used as control) and does not induce the degradation of Bcl-X L , XIAP, Bcl-2, Mcl-1, cIAP-1, IAP-2, or survivin. b The differences in the amount of LC3-II between samples in the presence and absence of chloroquine (CQ), representing the amount of LC3 that is degraded into autolysosomes, and allowing the measurement of the autophagic flux is represented. No additional LC3-II accumulation is observed when CP-31398 (48 h) is used in combination with CQ (2–24 h), showing that the autophagic flux induced by CP-31398 is blocked at the autophagosome-lysosome fusion step. c P62 expression level increases following 48 h treatment with CP-31398. d , e MDA-MB231 cells were transduced with LC3 fusion protein tagged with acid‑sensitive eGFP and acid‑insensitive RFP to monitor the specific loss of eGFP fluorescence upon acidification of the RFP + autophagosome following lysosomal fusion. Serum starvation induces autophagy with the loss of eGFP signal reflecting the fusion of autophagosomes with lysosomes. Fluorescence is seen from both eGFP and RFP channels after CP-31398 treatment, similarly to CQ that blocks autophagy through neutralization of lysosomal pH, demonstrating that CP-31398-induced autophagy flux is blocked at the autophagosomes/lysosomes fusion step. Dashed lines: plasma membrane. Scale bars: 10 μm ( c ). The percentage RFP + or RFP + /GFP + autophagosomes was evaluated in 100 cells after the indicated treatment ( d ). f Representative electron microscopy images of the autophagosomes containing undigested material presents in CP-31398 or chloroquine (CQ)-treated cells. Representative images of multivesicular bodies, mitochondria and endoplasmic reticulum are displayed as control. Data are representative of three independent experiments ( a – d , f ) or are the mean ± s.d. of three independent experiments ( e )

Article Snippet: From Merck: anti-p21 mouse mAb (clone OP64), anti-mdm2 mouse mAb (clone 2A10).

Techniques: Expressing, Transduction, Fluorescence, Neutralization, Electron Microscopy

Journal: Cancer Medicine

Article Title: Polymorphism rs2395655 affects LEDGF/p75 binding activity and p21WAF1/CIP1 gene expression in esophageal squamous cell carcinoma

doi: 10.1002/cam4.2067

Figure Lengend Snippet: Primers for RT‐PCR analysis and ChIP assay

Article Snippet: After preincubating in 10% goat serum for nonspecific binding block, slides were then incubated overnight at 4°C with mouse anti‐human p21 protein (BD Biosciences; 1:500 dilution).

Techniques: Sequencing

Journal: Cancer Medicine

Article Title: Polymorphism rs2395655 affects LEDGF/p75 binding activity and p21WAF1/CIP1 gene expression in esophageal squamous cell carcinoma

doi: 10.1002/cam4.2067

Figure Lengend Snippet: Reporter gene assay with constructs containing the pivotal region of the p21 promoter. (A) Schematic of four reporter gene constructs encompassing nucleotides −2308 to +204 of the p21 promoter, with sequences identical except for rs3829963 (–2119C/A) and rs2395655 (–809G/A) polymorphic sites. Luciferase expression in the four constructs in ESCC cells EC ‐109 (B) and KYSE 150 (C) were standardized by cotransfection with pRL ‐ SV 40. LEDGF /p75 expression vector was cotransfected to evaluate its potential effect on the activity of the p21 promoter, using pc DNA 3.0 empty vector as control. Fold increase was measured by defining the activity of the pGL 3‐Basic vector and pc DNA 3.0 empty vector as 1. The value of each reported construct was calculated as the mean fold increase ± SD from three independent experiments each performed in duplicate (top panel). LEDGF /p75 (p75 in brief) protein levels in EC ‐109 and KYSE 150 cells were examined by Western blot analysis (bottom panel). * P < 0.05 and ** P < 0.01 vs rs2395655 A allele‐containing counterparts, respectively

Article Snippet: After preincubating in 10% goat serum for nonspecific binding block, slides were then incubated overnight at 4°C with mouse anti‐human p21 protein (BD Biosciences; 1:500 dilution).

Techniques: Reporter Gene Assay, Construct, Luciferase, Expressing, Cotransfection, Plasmid Preparation, Activity Assay, Western Blot

Journal: Cancer Medicine

Article Title: Polymorphism rs2395655 affects LEDGF/p75 binding activity and p21WAF1/CIP1 gene expression in esophageal squamous cell carcinoma

doi: 10.1002/cam4.2067

Figure Lengend Snippet: LEDGF /p75 interacted with human genomic DNA sequence containing rs2395655 G allele. (A) The rs2395655 A>G transition created one cis‐regulatory element, that is STRE element ( AGGGGT , nucleotides −810 to −805), in the p21 promoter. Polymorphic sites of rs2395655 were italicized. (B) Nuclear extracts from KYSE 150 cells were used for electrophoretic mobility shift assay ( EMSA ) with two biotin‐labeled probes carrying rs2395655 G and A allele, respectively. Anti‐ LEDGF /p75 antibody (C‐16, 2 mg), IgG control antibody or a 400‐fold molar excess of cold probe was added to the binding reaction to examine the specificity of the protein‐ DNA complex formation. The black and hollow arrows indicated the shift and supershift bands, respectively. (C) Chromatin immunoprecipitation was performed using anti‐ LEDGF /p75 antibody (C‐16) and genomic DNA mixture of EC ‐109 and KYSE 410 cells, carrying rs2395655 GG and AA genotype, respectively. Human p21 region of interest was amplified and sequenced. Human GAPDH and FBXO 10 loci were included as respective negative and positive LEDGF /p75 binding controls

Article Snippet: After preincubating in 10% goat serum for nonspecific binding block, slides were then incubated overnight at 4°C with mouse anti‐human p21 protein (BD Biosciences; 1:500 dilution).

Techniques: Sequencing, Electrophoretic Mobility Shift Assay, Labeling, Binding Assay, Chromatin Immunoprecipitation, Amplification

Journal: Cancer Medicine

Article Title: Polymorphism rs2395655 affects LEDGF/p75 binding activity and p21WAF1/CIP1 gene expression in esophageal squamous cell carcinoma

doi: 10.1002/cam4.2067

Figure Lengend Snippet: LEDGF /p75 regulated the expression of p21 gene in ESCC cells with rs2395655 GG genotype. (A) Specific small interference RNA s (si‐p75‐1 and si‐p75‐2)‐mediated depletion of LEDGF /p75 (p75 in brief) induced obviously down‐regulated expression of p21 mRNA in EC ‐109 and KYSE 150 cells carrying rs2395655 GG genotype but not KYSE 410 and KYSE 450 cells with rs2395655 AA genotype. (B) Ectopic expression of p75 apparently increased the expression level of p21 mRNA in EC ‐109 and KYSE 150 cells, while no change of the p21 expression level was detected in KYSE 410 and KYSE 450 cells. For specific si RNA transfection and ectopic expression of p75, the mismatched si RNA (mock) and empty vector transfections were used as control, respectively. The relative signal intensity of each target gene was quantified and normalized to internal control, β‐actin. The graph indicates the relative mRNA expression levels of p75 and p21 and the values are mean ± SD for three independent experiments. M, DNA marker. Neg, negative control. * P < 0.01 and ** P > 0.05 vs controls, respectively

Article Snippet: After preincubating in 10% goat serum for nonspecific binding block, slides were then incubated overnight at 4°C with mouse anti‐human p21 protein (BD Biosciences; 1:500 dilution).

Techniques: Expressing, Transfection, Plasmid Preparation, Marker, Negative Control

Journal: Cancer Medicine

Article Title: Polymorphism rs2395655 affects LEDGF/p75 binding activity and p21WAF1/CIP1 gene expression in esophageal squamous cell carcinoma

doi: 10.1002/cam4.2067

Figure Lengend Snippet: Immunohistochemistry staining of the p21 protein in ESCC tissues. No‐anti p21, negative control with primary antibody replaced by PBS . Anti‐p21, the brown signals represented positive staining for p21 protein and the staining was scored on a scale as indicated in the Materials and Methods. Positive cases were defined as those with moderate to strong p21 nuclear staining in ≥ 10% of tumor cells. Magnification, × 100 (top panel) and × 400 (bottom panel), respectively

Article Snippet: After preincubating in 10% goat serum for nonspecific binding block, slides were then incubated overnight at 4°C with mouse anti‐human p21 protein (BD Biosciences; 1:500 dilution).

Techniques: Immunohistochemistry, Staining, Negative Control

Journal: Cancer Medicine

Article Title: Polymorphism rs2395655 affects LEDGF/p75 binding activity and p21WAF1/CIP1 gene expression in esophageal squamous cell carcinoma

doi: 10.1002/cam4.2067

Figure Lengend Snippet: Associations of clinical characteristics and genetic factors with the p21 protein expression of ESCC patients (n = 218)

Article Snippet: After preincubating in 10% goat serum for nonspecific binding block, slides were then incubated overnight at 4°C with mouse anti‐human p21 protein (BD Biosciences; 1:500 dilution).

Techniques: Expressing, Cell Differentiation, Variant Assay

Journal: Cancer Medicine

Article Title: Polymorphism rs2395655 affects LEDGF/p75 binding activity and p21WAF1/CIP1 gene expression in esophageal squamous cell carcinoma

doi: 10.1002/cam4.2067

Figure Lengend Snippet: Kaplan–Meier survival estimation of 218 ESCC patients related to possible predictors. (A) Elevated p21 protein expression indicated better postoperative outcome for ESCC patients (32.0 and 19.0 months of median survival for p21‐positive and negative patients, respectively, P = 0.001). (B) The median survival time of ESCC patients with rs2395655 GG genotype was significantly increased compared with rs2395655 AA or AG genotype (30.0 vs 20.0 months, P = 0.003)

Article Snippet: After preincubating in 10% goat serum for nonspecific binding block, slides were then incubated overnight at 4°C with mouse anti‐human p21 protein (BD Biosciences; 1:500 dilution).

Techniques: Expressing

Journal: Cancer Medicine

Article Title: Polymorphism rs2395655 affects LEDGF/p75 binding activity and p21WAF1/CIP1 gene expression in esophageal squamous cell carcinoma

doi: 10.1002/cam4.2067

Figure Lengend Snippet: Independent predictors of disease‐free survival time in multivariate analysis in ESCC patients (n = 218)

Article Snippet: After preincubating in 10% goat serum for nonspecific binding block, slides were then incubated overnight at 4°C with mouse anti‐human p21 protein (BD Biosciences; 1:500 dilution).

Techniques: Expressing, Variant Assay