nih3t3 murine embryo fibroblasts cell line  (ATCC)

Journal: Oncology Letters

Article Title: CDK4 has the ability to regulate Aurora B and Cenpp expression in mouse keratinocytes

doi: 10.3892/ol.2021.12993

Figure Lengend Snippet: CDK4 binds to regulatory regions of genes associated with chromosomal segregation. (A) ChIP analysis of NIH3T3 cells performed with an anti-CDK4 antibody (CDK4). Chromatin input and ChIP with anti-H3 antibody were used as positive controls, and normal IgG was used as a negative control. ‘Cont’ indicated the blank of the PCR reaction. The binding of CDK4 to specific promoters was analyzed by standard PCR with primers that amplified the regulatory sequences CENP-P, Aurkb, Ckap2, Zw10, Top2a and Mlf1ip genes. (B) Increased binding to the regulatory regions of CENP-P and Aurkb genes upon overexpression of CDK4 in NIH3T3 cells (NIH3T3-CDK4). ChIP analysis of NIH3T3 and NIH3T3-CDK4 cells was performed in three sequential immunoprecipitations with an anti-CDK4 antibody. H3, Histone 3; CDK4, cyclin-dependent kinase 4; ChIP, chromatin immunoprecipitation; CENP-P, Centromere Protein P; Aurkb, Aurora-B; Ckap2, cytoskeleton-associated protein 2; Zw10, centromere/kinetochore protein zw10 homolog; Top2a, DNA topoisomerase 2-a; Mlf1ip, MLF1-interacting protein.

Article Snippet: NIH3T3 murine embryo fibroblasts cell line was obtained from the American Type Culture Collection (Catalog number CRL-1658; ATCC).

Techniques: Negative Control, Binding Assay, Amplification, Over Expression, Chromatin Immunoprecipitation

Journal: Oncology Letters

Article Title: CDK4 has the ability to regulate Aurora B and Cenpp expression in mouse keratinocytes

doi: 10.3892/ol.2021.12993

Figure Lengend Snippet: Transcription and protein expression levels of CENP-P and Aurkb are increased following overexpression of CDK4. (A) Transcriptional levels of CENP-P, Aurkb, Zw10, Ckap2 and Top2a were determined by reverse transcription-quantitative PCR analysis. Shown are normalized expression ratios of cells overexpressing CDK4 (NIH3T3-CDK4 and 308-CDK4) compared with parental cells (NIH3T3 and 308). Values >1 denote increased transcriptional expression in CDK4 overexpressing cells, whereas values <1 denote reduced or equal transcription levels compared with parental cell lines. All the results were normalized with Gapdh expression. n=3 independent experiments, data are presented as the mean ± SEM. Student's t-test was performed. *P<0.05, **P<0.005 vs. appropriate parental cell line. (B) Western blot analysis of CENP-P, Aurkb and CDK4 in cells overexpressing CDK4 (NIH-CDK4, 308-CDK4) and the control cell lines infected with control retrovirus (NIH3T3, 308). β-Actin was used as a loading control. CDK4, cyclin-dependent kinase 4; CENP-P, Centromere Protein P; Aurkb, Aurora-B; Ckap2, cytoskeleton-associated protein 2; Zw10, centromere/kinetochore protein zw10 homolog; Top2a, DNA topoisomerase 2; MW, molecular weight.

Article Snippet: NIH3T3 murine embryo fibroblasts cell line was obtained from the American Type Culture Collection (Catalog number CRL-1658; ATCC).

Techniques: Expressing, Over Expression, Reverse Transcription, Real-time Polymerase Chain Reaction, Western Blot, Control, Infection, Molecular Weight