Basic Cell Culture Protocols

Pollard, Jeffrey W.; Walker, John M.

doi:10.1385/0896034410

Cited by 23 publications

(17 citation statements)

References 33 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Cell Culture-The RD1 (ATCC), HEK293 (ATCC), 10T1/2 (ATCC), and proliferating C2C12 (ATCC) cell lines were grown in DMEM supplemented with 10% fetal bovine serum (Hyclone) in a humidified CO 2 incubator at 37°C according to standard protocols (34). To induce differentiation in C2C12 cells and 10T1/2 cells, cells were grown to 70% confluence, and the medium was switched to DMEM supplemented with 2% horse serum (Hyclone).…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Myogenin Recruits the Histone Chaperone Facilitates Chromatin Transcription (FACT) to Promote Nucleosome Disassembly at Muscle-specific Genes

Lolis

Londhe

Beggs

et al. 2013

Journal of Biological Chemistry

View full text Add to dashboard Cite

Background:The FACT complex reorganizes nucleosomes and functions in DNA replication, transcription, and DNA repair. Results: Myogenin interacts with FACT, which is required for histone depletion at promoters and initiation of gene expression. Conclusion: Myogenin recruits the FACT complex to muscle-specific genes where FACT depletes nucleosomes at promoters, thus facilitating gene expression. Significance: The recruitment of FACT by myogenin is crucial to activate myogenin-dependent genes.

show abstract

Section: Methodsmentioning

confidence: 99%

“…Cell Transfections and Luciferase Assays-HEK293 and 10T1/2 cells were transiently transfected with calcium phosphate according to standard protocols (34). For the luciferase assay, the vectors pGL3 basic (Promega) and pGL3(ϩ) (Promega) were used as the negative and positive controls, respectively.…”

Section: Methodsmentioning

confidence: 99%

Myogenin Recruits the Histone Chaperone Facilitates Chromatin Transcription (FACT) to Promote Nucleosome Disassembly at Muscle-specific Genes

Lolis

Londhe

Beggs

et al. 2013

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…Proliferating C2C12 myoblasts (ATCC) were grown in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (HyClone) in a humidified CO 2 incubator at 37°C according to standard protocols (44). To induce differentiation into myotubes, cells were grown to 70% confluence and the medium was switched to DMEM supplemented with 2% horse serum (HyClone).…”

Section: Methodsmentioning

confidence: 99%

Gamma Interferon Modulates Myogenesis through the Major Histocompatibility Complex Class II Transactivator, CIITA

Londhe

Davie

2011

Molecular and Cellular Biology

View full text Add to dashboard Cite

Gamma interferon (IFN-␥) is an inflammatory cytokine that has complex effects on myogenesis. Here, we show that the IFN-␥-induced inhibition of myogenesis is mediated by the major histocompatibility complex (MHC) class II transactivator, CIITA, which binds to myogenin and inhibits its activity. In IFN-␥-treated myoblasts, the inhibition of muscle-specific genes includes the expression of myogenin itself, while in myotubes, myogenin expression is unaffected. Thus, CIITA appears to act by both repressing the expression and inhibiting the activity of myogenin at different stages of myogenesis. Stimulation by IFN-␥ in skeletal muscle cells induces CIITA expression as well as MHC class II gene expression. The IFN-␥-mediated repression is reversible, with myogenesis proceeding normally upon removal of IFN-␥. Through overexpression studies, we confirm that the expression of CIITA, independent of IFN-␥, is sufficient to inhibit myogenesis. Through knockdown studies, we also demonstrate that CIITA is necessary for the IFN-␥-mediated inhibition of myogenesis. Finally, we show that CIITA, which lacks DNA binding activity, is recruited to muscle-specific promoters coincident with reductions in RNA polymerase II recruitment. Thus, this work reveals how IFN-␥ modulates myogenesis and demonstrates a key role for CIITA in this process.Gamma interferon (IFN-␥) is an inflammatory cytokine that was first identified as an antiviral factor. IFN-␥ is a pleiotropic cytokine that regulates different immune responses and influences many physiological processes. Many studies have also shown that IFN-␥ influences skeletal muscle homeostasis and repair. Transient administration of exogenous IFN-␥ has been shown to improve healing of skeletal muscle and limit fibrosis (14). Endogenous IFN-␥ is required for efficient muscle regeneration, as mice lacking IFN-␥ show impaired muscle regeneration following cardiotoxin-induced damage (6). Expression of IFN-␥ is robust in proliferating C2C12 cells, but expression is diminished in differentiated C2C12 cells (6). Exogenous IFN-␥ influences the proliferation and differentiation of cultured myoblasts and appears to have a direct role on gene expression (25,26,28,48).Myoblasts have been shown to express immunological properties such as the complement component of both the classical and alternative pathways and major histocompatibility complex (MHC) genes. Exogenous IFN-␥ treatment has been shown to increase the expression of MHC class II genes, complement C components, intracellular adhesion molecule (Icam1), chemokine (C-C motif) ligand 5 (Ccl5; RANTES), chemokine (C-C motif) ligand 2 (Ccl2), and chemokine (C-X-C motif) ligand 10 (Cxcl10; Ip10) (15,25,28,48). It is not currently known how IFN-␥ mediates these transcriptional effects in myoblasts.The positive role for IFN-␥ established in muscle healing and repair suggests that this cytokine plays an important role in muscle biology. However, IFN-␥ signaling is likely to be tightly regulated, as negative effects of IFN-␥ have been observed as well. Whe...

show abstract

“…To determine plating efficiency, cells were seeded at a density of 50 000 cells cm 22 and the percentage of non-adhered cells was determined after 30, 60 and 120 min and 24 h. To determine growth response, cells were seeded at 10 000 cells cm 22 , trypsinized at 3, 7, 10, 14, 17, 21, 24 and 28 days, and counted using a haemocytometer. Population doublings were calculated as described previously (Pollard & Walker, 1997). Serum dependency was assessed by seeding cells at 1000 cells cm…”

mentioning

confidence: 99%

“…For karyotyping, cells at exponential growth phase at subculture 20 were incubated overnight at 30 uC with 0.5 mg demecolcine ml 21 (Sigma-Aldrich) and chromosomes were prepared as described previously (Pollard & Walker, 1997). Immunohistochemistry was used to confirm that cells in culture were fibroblasts or keratinocytes.…”

mentioning

confidence: 99%

In vitro biology of fibropapilloma-associated turtle herpesvirus and host cells in Hawaiian green turtles (Chelonia mydas)

Work

Dagenais

Balazs

et al. 2009

Journal of General Virology

View full text Add to dashboard Cite

Fibropapillomatosis (FP) of green turtles has a global distribution and causes debilitating tumours of the skin and internal organs in several species of marine turtles. FP is associated with a presently non-cultivable alphaherpesvirus Chelonid fibropapilloma-associated herpesvirus (CFPHV). Our aims were to employ quantitative PCR targeted to pol DNA of CFPHV to determine (i) if DNA sequesters by tumour size and/or cell type, (ii) whether subculturing of cells is a viable strategy for isolating CFPHV and (iii) whether CFPHV can be induced to a lytic growth cycle in vitro using chemical modulators of replication (CMRs), temperature variation or co-cultivation. Additional objectives included determining whether non-tumour and tumour cells behave differently in vitro and confirming the phenotype of cultured cells using cell-type-specific antigens. CFPHV pol DNA was preferentially concentrated in dermal fibroblasts of skin tumours and the amount of viral DNA per cell was independent of tumour size. Copy number of CFPHV pol DNA per cell rapidly decreased with cell doubling of tumour-derived fibroblasts in culture. Attempts to induce viral replication in known CFPHV-DNA-positive cells using temperature or CMR failed. No significant differences were seen in in vitro morphology or growth characteristics of fibroblasts from tumour cells and paired normal skin, nor from CFPHV pol-DNA-positive intestinal tumour cells. Tumour cells were confirmed as fibroblasts or keratinocytes by positive staining with antivimentin and anti-pancytokeratin antibodies, respectively. CFPHV continues to be refractory to in vitro cultivation.

show abstract

Basic Cell Culture Protocols

Cited by 23 publications

References 33 publications

Myogenin Recruits the Histone Chaperone Facilitates Chromatin Transcription (FACT) to Promote Nucleosome Disassembly at Muscle-specific Genes

Myogenin Recruits the Histone Chaperone Facilitates Chromatin Transcription (FACT) to Promote Nucleosome Disassembly at Muscle-specific Genes

Gamma Interferon Modulates Myogenesis through the Major Histocompatibility Complex Class II Transactivator, CIITA

In vitro biology of fibropapilloma-associated turtle herpesvirus and host cells in Hawaiian green turtles (Chelonia mydas)

Contact Info

Product

Resources

About