Journal: Genome Medicine

Article Title: Integrative epigenomic and high-throughput functional enhancer profiling reveals determinants of enhancer heterogeneity in gastric cancer

doi: 10.1186/s13073-021-00970-3

Figure Lengend Snippet: Cis -analysis of differential enhancers. a Distribution of enhancers associated with copy number. CN-abnormal enhancers represent differential enhancers with altered DNA copy numbers in OCUM1 or SNU16. CN-associated enhancers represent differential enhancers showing concordant changes in H3K27ac signals and DNA copy number. MYC , WDR11 , FGFR2 , CD44 , PDHX , and ING1 -associated enhancers are highlighted as they show a statistically significant correlation between enhancer copy numbers with target gene expression levels. b Correlation of H3K27ac signals and ING1 gene expression with the DNA copy numbers for the ING1 enhancer among multiple GC lines. c H3K27ac ChIP-seq and CapSTARR-seq tracks at the enhancer region harboring the ARL4C -associated SNP rs1464264 in OCUM1 and SNU16 cells. d H3K27ac ChIP-seq tracks in GC lines and pcHi-C associations in gastric tissues. The SNP rs1464264 genotype is annotated above the H3K27ac ChIP-seq track in each cell line. Significant chromatin interactions are shown below the axis (green loop). e Differences in expression of ARL4C in three groups of cell lines with different SNP rs1464264 genotypes (GG, AG, and AA). *: P < 0.05, ns: not significant, Mann–Whitney U test. f Difference in expression of ARL4C in three groups of GC patients with different SNP rs1464264 genotypes (GG, AG, and AA) in the Singapore cohort (n= 161). *: P < 0.05, ns: not significant, Student’s t test. g Survival analysis comparing patient groups with samples exhibiting low (green) and high (red) expression of ARL4C in the ACRG cohort ( n = 300). P value is calculated using the Log-rank test. Survival data are indicated for every 25 months

Article Snippet: The following antibodies were used: ARL4C (#10202-1-AP, Proteintech), ING1 (#14625, Cell Signalling Technology) and GAPDH (#60004-1-Ig, Proteintech).

Techniques: Expressing, ChIP-sequencing, MANN-WHITNEY

Journal: Cancer Cell International

Article Title: p33ING1b regulates acetylation of p53 in oral squamous cell carcinoma via SIR2

doi: 10.1186/s12935-020-01489-0

Figure Lengend Snippet: ING1b regulates p53-acetylation and downstream activation of pro-apoptotic pathway. a Relative protein expression of p53, acetylated p53 (at Lys382) and ING1b in OSCC cell lines YD-9 (wild type TP53 ), YD-8 ( TP53 point mutation), and YD-38 ( TP53 deletion). GAPDH was used as a loading control. Data is representative of three independent experiments. b Immunoblot analysis of total and acetylated p53 (at Lys382), and pro-apoptotic Bax and p21 in YD-9 and YD-8 cells stably transduced with control or INGb1 shRNA shows ING1b regulates acetylation and downstream expression of pro-apoptotic proteins. GAPDH was used as a loading control. Data is representative of three independent experiments. Numbers below the figure shows relative expression of ING1b as determined by densitometry analysis. c Cell proliferation was assayed for 3 days in YD-9 and YD-8 cells transiently transfected with control or ING1b shRNA, 48 h after transfection. Data represented in from 3 different independent experiments. Error bars, SD

Article Snippet: Antibodies (all antibodies were used at 1:1000 dilution) used were p53, Acetyl-p53(K382), ING1b, Bax, SIR2 (SIRT1), p21, Cleaved Caspase-3, Bcl-xL, and GAPDH (Cell Signaling, Cambridge, MA, USA).

Techniques: Activation Assay, Expressing, Mutagenesis, Western Blot, Stable Transfection, Transduction, shRNA, Transfection

Journal: Cancer Cell International

Article Title: p33ING1b regulates acetylation of p53 in oral squamous cell carcinoma via SIR2

doi: 10.1186/s12935-020-01489-0

Figure Lengend Snippet: Over expression of ING1b increase acetylated p53, decrease cell proliferation, and induce apoptosis. a Immunoblot analysis of ING1b, total and acetylated p53 (at Lys382), and pro-apoptotic Bax and p21 in YD-9 and YD-8 cells transiently transfected with control or ING1 expression plasmid. GAPDH was used as a loading control. Data is representative of three independent experiments. Numbers below the figure shows relative expression of ING1b as determined by densitometry analysis. b Cell proliferation was assayed for 3 days in YD-9 and YD-8 cells transiently transfected with control or ING1 expression plasmid, 48 h after transfection. Data represented in from 3 different independent experiments. Error bars, SD. c Immunoblot analysis of anti-apoptotic Bcl-XL and pro-apoptotic Cleaved Caspase-3 in YD-8 and YD-9 cells transiently transfected with control or ING1b expression plasmids. GAPDH was used as a loading control. Data is representative of three independent experiments. d Immunoblot analysis of ING1b, total and acetylated p53 (at Lys382), anti-apoptotic Bcl-XL and pro-apoptotic Cleaved Caspase-3 in Ca9-22 and Sa-3 cell lines transiently transfected with control or ING1b expression plasmids. GAPDH was used as a loading control. Data is representative of three independent experiments

Article Snippet: Antibodies (all antibodies were used at 1:1000 dilution) used were p53, Acetyl-p53(K382), ING1b, Bax, SIR2 (SIRT1), p21, Cleaved Caspase-3, Bcl-xL, and GAPDH (Cell Signaling, Cambridge, MA, USA).

Techniques: Over Expression, Western Blot, Transfection, Expressing, Plasmid Preparation

Journal: Cancer Cell International

Article Title: p33ING1b regulates acetylation of p53 in oral squamous cell carcinoma via SIR2

doi: 10.1186/s12935-020-01489-0

Figure Lengend Snippet: ING1b expression is correlated to p53-mediated p21 and Bax transcriptional activity. a Firefly luciferase promoter reporters for CDKN1A (encoding p21) or BAX and Renilla luciferase control reporters were co-transfected in YD-9 and YD-8 cells 48 h post-transfection with either control or ING1b expression plasmid. Dual luciferase assay was performed 24 h post-transfection of luciferase reporters. Data was normalized to Renilla luciferase. Error bars, SD. *P < 0.05. b Firefly luciferase promoter reporters for CDKN1A (encoding p21) or BAX and Renilla luciferase control reporters were co-transfected in YD-9 and YD-8 cells stably transduced with control or ING1b shRNA. Dual luciferase assay was performed 24 h post-transfection of luciferase reporters. Data was normalized to Renilla luciferase. Error bars, SD. *p < 0.05. c Chromatin immunoprecipitation assay (ChIP) shows direct binding of acetylated-p53 to both CDKN1A and BAX promoters, which was significantly more following transient overexpression of ING1b. Error bars, SD. *p < 0.05. d ChIP assay performed with primers specific to downstream of promoters of CDKN1A and BAX did not show any enrichment and was used as a control to rule out false-positive in results obtained in c due to incomplete DNA fragmentation. Error bars, SD. e ChIP confirmed ING1b mediates p53-mediated transcriptional activation of CDKN1A and BAX . Significant attenuation of direct interaction with either promoter was observed in YD-9 and YD-8 cells stably transduced with ING1b shRNA. Error bars, SD. *pP < 0.05. f ChIP assay performed with primers specific to downstream of promoters of CDKN1A and BAX did not show any enrichment and was used as a control to rule out false-positive in results obtained in e due to incomplete DNA fragmentation. Error bars, SD

Article Snippet: Antibodies (all antibodies were used at 1:1000 dilution) used were p53, Acetyl-p53(K382), ING1b, Bax, SIR2 (SIRT1), p21, Cleaved Caspase-3, Bcl-xL, and GAPDH (Cell Signaling, Cambridge, MA, USA).

Techniques: Expressing, Activity Assay, Luciferase, Transfection, Plasmid Preparation, Stable Transfection, Transduction, shRNA, Chromatin Immunoprecipitation, Binding Assay, Over Expression, Activation Assay

Journal: Cancer Cell International

Article Title: p33ING1b regulates acetylation of p53 in oral squamous cell carcinoma via SIR2

doi: 10.1186/s12935-020-01489-0

Figure Lengend Snippet: ING1b upregulates acetylated-p53 by modulating SIR2 levels. a Immunoblot analysis of ING1b, SIR2, acetylated and total p53 in YD-9 and YD-8 cells either transiently overexpressing ING1b alone or along with SIR2 or stably transduced with ING1b shRNA. GAPDH was used as a loading control. Data is representative of three independent experiments. b Effect of ING1b on p53 acetylation is mediated via SIR2. Immunoblot analysis of ING1b, SIR2, and acetylated p53 in YD-9 and YD-8 cells stably transduced with control shRNA, ING1b shRNA, or both ING1b and SIR2 shRNA. GAPDH was used as a loading control. Data is representative of three independent experiments. c Quantitative real-time PCR analysis of relative expression of SIR2 mRNA in YD-9 and YD-8 cells in which ING1b is overexpressed or knocked down. Data shown was normalized to GAPDH mRNA expression and expressed relative to parental YD-9 or YD-8 cells, respectively. Error bars, SD. *P < 0.05

Article Snippet: Antibodies (all antibodies were used at 1:1000 dilution) used were p53, Acetyl-p53(K382), ING1b, Bax, SIR2 (SIRT1), p21, Cleaved Caspase-3, Bcl-xL, and GAPDH (Cell Signaling, Cambridge, MA, USA).

Techniques: Western Blot, Stable Transfection, Transduction, shRNA, Real-time Polymerase Chain Reaction, Expressing

Journal: Cancer Cell International

Article Title: p33ING1b regulates acetylation of p53 in oral squamous cell carcinoma via SIR2

doi: 10.1186/s12935-020-01489-0

Figure Lengend Snippet: Synergistic effect of ING1b and SIR2 on chemosensitivity of YD-9 and YD-8 cells to cisplatin. Parental YD-9 a or YD-8 b cells, or cells transiently transfected with ING1b expression plasmid, stably transduced with ING1b shRNA, stably transduced with SIR2 shRNA or cells overexpressing ING1b and in which SIR2 has been stably knocked down, were treated with 5 µM of cisplatin and cell viability was measured after indicated times. Same as a and b , but YD-9 cells c or YD-8 cells d were treated with indicated concentrations of cisplatin for 12 h. All data is representative of three independent experiments, each done in triplicate. Error bars, SD. *P < 0.05

Article Snippet: Antibodies (all antibodies were used at 1:1000 dilution) used were p53, Acetyl-p53(K382), ING1b, Bax, SIR2 (SIRT1), p21, Cleaved Caspase-3, Bcl-xL, and GAPDH (Cell Signaling, Cambridge, MA, USA).

Techniques: Transfection, Expressing, Plasmid Preparation, Stable Transfection, Transduction, shRNA