murine embryo fibroblast cell line nih 3t3  (ATCC)

Journal: bioRxiv

Article Title: True-to-scale DNA-density maps correlate with major accessibility differences between active and inactive chromatin

doi: 10.1101/2022.03.23.485308

Figure Lengend Snippet: High-resolution, true-to-scale DNA density landscape of human fibroblast nucleus a Voronoi-tessellated SMLM-fBALM image of a mid-optical section (thickness ∼100 nm through a diploid human fibroblast cell nucleus (BJ1) stained with Sytox Orange (see Methods for further explanation how this image was generated). The color code indicates the estimated range of 3D DNA densities from <5 Mbp/µm 3 to >40 Mbp/µm 3 , reflecting 2D SMP densities <2,500 SMPs/µm 2 to >20,000 SMPs/µm 2 (see and Results for 3D DNA densities estimated from 2D SMP densities in 100 nm thick sections). b, 1-3 Magnifications of the three boxed areas in (a) demonstrate sites located at the nuclear periphery (1) and in the nuclear interior (2, 3); edge length 3000 nm. Asterisks denote IC- lacunae. b, 1’–3’ Further magnification of boxed areas in (1-3), edge lengths 1000 nm (1’), and 500 nm (2’,3’). c, 1’’–3’’ upper row: Further magnification of boxed areas in (1’-3’), edge length 200 nm. lower row: Individual Voronoi centers (seeds) are shown for the same Voronoi areas as in 1’’ – 3’’. d, 1’’’–3’’’ same areas as in c 1’’ depicted here with another color code (shown at bottom) for 3D DNA densities from 40 Mbp/µm 3 to about 350 Mbp/µm 3 , reflecting a range of 2D SMP densities between ∼20,000 and ∼180,000 SMPs/µm 2 .

Article Snippet: h-TERT BJ1 cells, immortalized, diploid human cell line from human foreskin fibroblasts (ATCC #CRL-2522), HeLa cells, a human cervix carcinoma derived cell line with a near-triploid karyotype (n = 68-70 ), and C3H 10T1/2 cells, derived from murine embryo fibroblast cell line (ATCC #CCL-226) were cultured in DMEM with 5% CO2 at 37 °C.

Techniques: Staining, Generated

Journal: bioRxiv

Article Title: True-to-scale DNA-density maps correlate with major accessibility differences between active and inactive chromatin

doi: 10.1101/2022.03.23.485308

Figure Lengend Snippet: Comparison of high-resolution DNA landscapes of mouse C3H T1/2 fibroblast nuclei recorded with SMLM and ESI a 100 nm optical mid-section through a mouse C3H T1/2 fibroblast nucleus recorded with SMLM-fBALM. b-e ESI micrographs taken from acrylic 100 nm thin mid-sections through another C3H T1/2 fibroblast nucleus. b and d show superimposed elemental maps of phosphorus (green) and nitrogen (red). c and e present the phosphorus map only, color-coded for different phosphorus densities as a reflection of DNA densities. For better comparison, the same color-coded look-up table (jet) was used for SMLM (a) and ESI images (c,e; blue indicates lowest DNA densities that belong to the IC, lined by light blue and green colored intermediate densities (PR); red marks high DNA compaction attributed to the INC (compare and ). Arrows in (d) and (e) indicate examples of chromocenters. In order to secure that only the DNA landscape was recorded, RNA was digested in both nuclei prior to imaging.

Article Snippet: h-TERT BJ1 cells, immortalized, diploid human cell line from human foreskin fibroblasts (ATCC #CRL-2522), HeLa cells, a human cervix carcinoma derived cell line with a near-triploid karyotype (n = 68-70 ), and C3H 10T1/2 cells, derived from murine embryo fibroblast cell line (ATCC #CCL-226) were cultured in DMEM with 5% CO2 at 37 °C.

Techniques: Comparison, Imaging

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