Journal: Scientific Reports

Article Title: Species-specific mutual regulation of p53 and miR-138 between human, rat and mouse

doi: 10.1038/srep26187

Figure Lengend Snippet: ( A ) Western blot analyses were performed to examine the effect of p53 siRNA on endogenous p53 protein levels in H460 cells (top); qRT-PCR assays were used to examine miR-138 and pri-miR-138 levels in H460 cells treated with p53 siRNA for 48 h (bottom). ( B ) Western blot analyses were performed to examine phosphorylated p53 protein levels in H460 cells treated with 2 Gy of ionizing radiation for the indicated times (0 and 4 hours) (top). Quantitative RT-PCR assays were performed to examine miR-138, pri-miR-138-1 and pri-miR-138-2 levels in H460 cells treated with 2 Gy of ionizing radiation for the indicated times (0, 2, 4, 8 and 16 hours) (bottom). U6 snRNA served as an internal control for miRNAs; GAPDH mRNA was used for normalization of pri-miRNA levels. ( C ) Western blot analyses were performed to examine p53 protein levels in H460 and H1299 cells (top); quantitative RT-PCR assays were performed to examine miR-138, pri-miR-138-1 and pri-miR-138-2 levels in H460 and H1299 cells treated with different doses (0 and 2 Gy) of ionizing radiation for the indicated time (4 hour) (bottom). ( D ) Western blot analyses were performed to examine p53 protein levels in H1299 cells transfected with pRC/CMV or pRC/p53 plasmids for 48h (top). Quantitative RT-PCR assays were performed to examine miR-138, pri-miR-138-1 and pri-miR-138-2 levels in H1299 cells transfected with pRC/CMV or pRC/p53 plasmids for 48h (bottom). Results are expressed as means ± s.d. for three independent experiments. * P < 0.05; ** P < 0.01 versus control.

Article Snippet: Recombinant p53 protein was purchased from OriGene (OriGene Technologies, Rockville, MD, USA).

Techniques: Western Blot, Quantitative RT-PCR, Transfection

Journal: Scientific Reports

Article Title: Species-specific mutual regulation of p53 and miR-138 between human, rat and mouse

doi: 10.1038/srep26187

Figure Lengend Snippet: ( A ) Schematic illustration showing that pre-miR-138-1 and pre-miR-138-2 are both intergenic. A putative p53 binding site (BS) located −4285 to −4263 bp upstream of miR-138-1; a predicted p53 binding site located downstream (1569 to 1599 bp) of miR-138-2. ( B ) ChIP-qPCR analyses were preformed using digested chromatin from H460 cells. The predicted p53 binding region of miR-138 was amplified from immunoprecipitates of anti-p53, anti-Histone H3 or normal rabbit IgG control. The negative control primer for a “promoter free” region in the genome to monitor the background noise; hTERT primer was used as a positive control. The fold enrichment over the IgG control is represented (mean ± s.d.; n = 3; ** P < 0.01). ( C ) p53 binds to the predicted binding site (BS) for miR-138 in vitro . Electrophoretic mobility shift assay (EMSA) was performed with 100 ng p53 wt protein and biotin-labelled oligonucleotides. The p53 antibody and unlabeled competitor probe were added as indicated. ( D ) The mutant p53-BS probes (top) were used in the EMSA with the p53 wt protein (bottom).

Article Snippet: Recombinant p53 protein was purchased from OriGene (OriGene Technologies, Rockville, MD, USA).

Techniques: Binding Assay, Amplification, Negative Control, Positive Control, In Vitro, Electrophoretic Mobility Shift Assay, Mutagenesis

Journal: Scientific Reports

Article Title: Species-specific mutual regulation of p53 and miR-138 between human, rat and mouse

doi: 10.1038/srep26187

Figure Lengend Snippet: ( A ) H460 (p53+/+) cells were co-transfected with luciferase reporter plasmids pGL4-138-1, pGL4-138-2 or empty pGL4-basic vector, with p53 siRNA or a control siRNA. After 48 h, the luciferase activity was measured. ( B ) H1299 (p53−/−) cells were co-transfected with luciferase reporter plasmids pGL4-138-1, pGL4-138-2 or empty pGL4-basic vector with pRC/p53, pRC/p53ΔTA or empty pRC/CMV plasmid. After 48 h, the luciferase activity was measured. ( C ) H1299 (p53−/−) cells were co-transfected with luciferase reporter plasmids pGL4-138-1, pGL4-138-2 or empty pGL4-basic vector with pRC/p53, pCMV/p63, pCMV/p73 or control pRC/CMV plasmid. After 48 h, the luciferase activity was measured. ( D ) H1299 (p53−/−) cells were transfected with pRC/p53, pCMV/p63, pCMV/p73 or control pRC/CMV plasmid. After 48 h, quantitative RT-PCR was performed to examine miR-138 and pri-miR-138 levels. Data are representative of at least three independent experiments (means ± s.d.). * P < 0.05; ** P < 0.01 versus control.

Article Snippet: Recombinant p53 protein was purchased from OriGene (OriGene Technologies, Rockville, MD, USA).

Techniques: Transfection, Luciferase, Plasmid Preparation, Activity Assay, Quantitative RT-PCR

Journal: Scientific Reports

Article Title: Species-specific mutual regulation of p53 and miR-138 between human, rat and mouse

doi: 10.1038/srep26187

Figure Lengend Snippet: (A,B) Western blot analyses were performed to examine the effects of p53 siRNA on endogenous p53 protein levels in NIH/3T3 and H9C2 cells (top); quantitative RT-PCR assays were performed to examine miR-138 and pri-miR-138 levels in two cell types (NIH/3T3 and H9C2) treated with p53 siRNA for 48 h. ( C ) The putative p53 binding sites loacted upstream or downstream 5 kb of miR-138-1 or miR-138-2 in human, mouse and rat. ( D ) ChIP-qPCR analysis were performed using digested chromatin from H460, NIH/3T3 and H9C2 cells. The predicted p53 binding region of miR-138 was amplified from immunoprecipitates of anti-p53, anti-Histone H3 or normal rabbit IgG control.

Article Snippet: Recombinant p53 protein was purchased from OriGene (OriGene Technologies, Rockville, MD, USA).

Techniques: Western Blot, Quantitative RT-PCR, Binding Assay, Amplification

Journal: Scientific Reports

Article Title: Species-specific mutual regulation of p53 and miR-138 between human, rat and mouse

doi: 10.1038/srep26187

Figure Lengend Snippet: ( A ) Summary of the reported miRNAs targeting p53. ( B ) qRT-PCR assays were performed to examine the effects of miR-138 mimic transfection on p53 gene transcription (GAPDH served as an internal control). ( C ) Alignment of the miR-138 sequence. MiR-138 sequences from three species of mammals (Human, Mouse and Rat) were compared using ClustalW server ( http://www.ebi.ac.uk/Tools/msa/clustalw2/ ). ( D ) Alignment of p53 amino acid sequences and p53 3′ UTR nucleotide sequences. ( E ) The predicted miR-138 targeting sequence located in the 3′ UTR of the p53 mRNA of rat and mouse. ( F ) Human p53 mRNA 3′ UTR lacks miR-138 targeting sequence, with a three-base difference. ( G ) Human p53 mRNA 3′ UTR (NM_001126114) was mutated to contain an miR-138 targeting sequence (left); dual luciferase reporter assays were performed to test the interaction of miR-138 with the wild-type predicted p53 3′ UTR targeting sequences (H p53 wt-3′ UTR) and the mutated targeting sequences (H p53-mut-3′ UTR). * P < 0.05 versus control. Data are representative of at least three independent experiments (means ± SD).

Article Snippet: Recombinant p53 protein was purchased from OriGene (OriGene Technologies, Rockville, MD, USA).

Techniques: Quantitative RT-PCR, Transfection, Sequencing, Luciferase

Journal: Scientific Reports

Article Title: Species-specific mutual regulation of p53 and miR-138 between human, rat and mouse

doi: 10.1038/srep26187

Figure Lengend Snippet: ( A ) The predicted miRNAs targeting sequences located in the 3′ UTR of the p53 mRNA of human, rat and mouse. The 3′UTR of human, mouse and rat p53 mRNA all have miR-485 conserved predicted target sites, but only the 3′UTRs of human p53 mRNA has miR-125b target site, ( B–D ) Quantitative RT-PCR assays were performed to examine p53 mRNA levels in three cell types (H460, NIH/3T3 and H9C2) treated with p53 siRNA and other miRNAs for 48 h. GAPDH served as the internal control (top). Western blot analyses were performed to examine the effects of p53 siRNA and miRNAs on endogenous p53 protein levels in H460, NIH/3T3 and H9C2 cells (bottom).

Article Snippet: Recombinant p53 protein was purchased from OriGene (OriGene Technologies, Rockville, MD, USA).

Techniques: Quantitative RT-PCR, Western Blot

Journal: Scientific Reports

Article Title: Species-specific mutual regulation of p53 and miR-138 between human, rat and mouse

doi: 10.1038/srep26187

Figure Lengend Snippet: ( A,B ) Two normal cell lines (human normal breast cell HBL-100 and normal liver cell L02) were transfected with control or p53 siRNA. After treament for 24 h, cells were transfected with pRC/CMV or pRC/P53 plasmids (4 μg/well in 6-well plates) for another 48 h. Western blot analyses were performed to examine p53 protein levels (top); quantitative RT-PCR assays were performed to examine miR-138 and p53 levels (bottom). ( C ) Quantitative RT-PCR assays were performed to examine the effects of miR-138 mimic transfection on p53 gene transcription in HBL-100 and L02 cells (GAPDH served as an internal control). ( D,E ) Quantitative RT-PCR assays were performed to examine miR-138 and p53 relative levels in human and rat tissues.

Article Snippet: Recombinant p53 protein was purchased from OriGene (OriGene Technologies, Rockville, MD, USA).

Techniques: Transfection, Western Blot, Quantitative RT-PCR