Journal:

Article Title: The role of acetylation in rDNA transcription

doi:

Figure Lengend Snippet: TSA, an inhibitor of histone deacetylation, stimulates rDNA transcription in transient transfection assays. NIH 3T3 cells were transfected with 1 µg rDNA reporter (pSMECAT) for 5 h and then treated with the indicated concentrations of TSA. After 19 h, cell lysates were prepared and assayed for CAT activity as described in Materials and Methods. Results were visualized and quantified using a Molecular Dynamics PhosphorImager. (A) Image of a CAT assay from a typical experiment. (B) Graph depicting the effect of TSA on the expression of pSMECAT (means ± SD, n = 12).

Article Snippet: Tissue culture and cell lines NIH 3T3 cells (ATCC no. CCL163) were obtained from the ATCC.

Techniques: Transfection, Activity Assay, Expressing

Journal:

Article Title: The role of acetylation in rDNA transcription

doi:

Figure Lengend Snippet: TSA stimulates transcription from the rRNA genes. Nuclei were isolated from NIH 3T3 cells treated for 24 h with the indicated concentrations of TSA and rDNA transcription was measured by nuclear run-on assay as described in Materials and Methods. In vitro labeled RNA was isolated using TRIzol reagent and purified by ethanol precipitation and/or Sephadex G-25 columns. The transcripts were hybridized to a fragment of the mouse rDNA promoter (–168 to +292) (10 µg DNA) immobilized on Zeta-Probe membrane. The results were visualized with a Molecular Dynamics PhosphorImager. (Upper) The results of a typical experiment. (Lower) The results from repeated experiments were quantitated and plotted relative to the control. Each point represents the mean ± SD (n = 7).

Article Snippet: Tissue culture and cell lines NIH 3T3 cells (ATCC no. CCL163) were obtained from the ATCC.

Techniques: Isolation, Nuclear Run-on Assay, In Vitro, Labeling, Purification, Ethanol Precipitation, Membrane, Control

Journal:

Article Title: The role of acetylation in rDNA transcription

doi:

Figure Lengend Snippet: TSA treatment results in accumulation of acetylated histone H4 in rDNA chromatin. Analysis of DNA immunoprecipitated with anti-acetyl-histone H4 antibody (Upstate Biotechnology) from chromatin isolated from TSA-treated or control NIH 3T3 cells. Cells were crosslinked in situ, collected and lysed. Resulting protein–DNA complexes were sheared by sonication and immunoprecipitated with antibody to acetylated histone H4 as described in Materials and Methods. After recovery of DNA was determined by spectrophotometry, recovery of rDNA was determined by PCR or dot blotting. (A) Scheme of PCR primers used in these experiments and the rDNA promoter. The resulting size of each product is indicated. (B) Nested PCR analysis of ChIP DNA from control and TSA-treated cells. The first round of PCR was performed with primer pair 3 and 4 and the second round with primer pair 1 and 2. The 350 bp final product is shown. (C) Three independent PCR/ChIP experiments similar to those described in (B) were visualized and quantitated using a Molecular Dynamics scanning densitometer. The results are expressed relative to the level of PCR product in the control sample (means ± SD). (D) Hybridization analysis of ChIP DNA immunoprecipitated from control and TSA-treated cells. After the DNA was immunoprecipitated and purified, it was immobilized on Zeta Probe membranes. The membranes were probed with a 400 bp fragment (PCR primers 3 and 4) synthesized in the presence of [α-32P]dCTP.

Article Snippet: Tissue culture and cell lines NIH 3T3 cells (ATCC no. CCL163) were obtained from the ATCC.

Techniques: Immunoprecipitation, Isolation, Control, In Situ, Sonication, Spectrophotometry, Nested PCR, Hybridization, Purification, Synthesized

Journal:

Article Title: The role of acetylation in rDNA transcription

doi:

Figure Lengend Snippet: TSA does not reverse the inhibition of rDNA transcription by Rb in transient transfection assays. NIH 3T3 cells were transfected with 2 µg rDNA reporter (pSMECAT) and 2 µg DNA of an Rb expression plasmid or empty vector (pCMV5) for 5 h and then treated with the indicated concentrations of TSA. After 19 h, cell lysates were prepared and assayed for CAT activity as described in Materials and Methods. Results were visualized and quantitated using a Molecular Dynamics PhosphorImager. (A) CAT assays from a typical experiment. (B) Three independent experiments similar to those described in (A) were quantitated, adjusted for protein in the assay and expressed as a fraction of CAT activity (± SEM) observed in the control sample.

Article Snippet: Tissue culture and cell lines NIH 3T3 cells (ATCC no. CCL163) were obtained from the ATCC.

Techniques: Inhibition, Transfection, Expressing, Plasmid Preparation, Activity Assay, Control

Journal:

Article Title: The role of acetylation in rDNA transcription

doi:

Figure Lengend Snippet: Overexpression of histone acetyltransferases stimulates rDNA transcription in transient transfection assays. NIH 3T3 cells were transfected with 1 µg rDNA reporter (pSMECAT) and different amounts (0.5, 1.0 or 2.0 µg DNA) of p300, PCAF, PCAF delHAT or CBP expression plasmids (all in pCDNA3.1). After 24 h, cell lysates were prepared and assayed for CAT activity. The results were visualized, normalized and quantitated using a Molecular Dynamics PhosphorImager. (Upper) CAT assays from a typical experiment. (Lower) A graph summarizing the CAT assays from three experiments (means ± SD). The results were normalized to the results obtained with pCAF delHAT.

Article Snippet: Tissue culture and cell lines NIH 3T3 cells (ATCC no. CCL163) were obtained from the ATCC.

Techniques: Over Expression, Transfection, Expressing, Activity Assay

Journal: PLOS Pathogens

Article Title: Eph receptor tyrosine kinases are functional entry receptors for murine gammaherpesvirus 68

doi: 10.1371/journal.ppat.1013263

Figure Lengend Snippet: (A) Schematic of Eph receptor-dependent block of MHV68 infection of susceptible cell lines (BioRender.com/jdlijfo). (B) Normalized read counts of the 14 Eph receptor genes in NIH 3T3 cells (GEO dataset series GSE196318 ). (C) Dose-dependent inhibition of MHV68 infection by soluble murine EphA4-Fc and EphB3-Fc but not EphA6-Fc on NIH 3T3 murine fibroblasts. MHV68 ORF59-GFP was pre-incubated with murine EphA4-Fc, EphA6-Fc or EphB3-Fc. EphA2-Fc and PBS were used as controls. GFP expression was measured by flow cytometry. Infection is indicated as percentage of GFP + cells normalized to PBS controls. Mean of three independent experiments, error bars represent SD. (D-G) Cell type-dependent inhibition of MHV68 infection by soluble murine Eph proteins at 100 nM homodimerized protein. EphA2-Fc and PBS were used as controls. GFP expression as indicator of infection was measured by flow cytometry. Infection is shown as percentage of GFP + cells normalized to PBS controls. Mean and symbols represent three individual experiments, error bars represent SD. Statistical significance was evaluated by ordinary one-way ANOVA, corrected by Holm-Šídák’s multiple comparisons test. *: p-value < 0.05, ***: p-value < 0.001, ****: p-value < 0.0001.

Article Snippet: NIH 3T3 (RRID:CVCL_0594) and NIH 3T12 (ATCC CCL-164, Manassas, VA) murine fibroblasts were maintained in DMEM supplemented with 8% FCS, 100 IU/mL penicillin, 100 μg/mL streptomycin, and 2 mM L-glutamine (D8).

Techniques: Blocking Assay, Infection, Inhibition, Incubation, Expressing, Flow Cytometry

Journal: PLOS Pathogens

Article Title: Eph receptor tyrosine kinases are functional entry receptors for murine gammaherpesvirus 68

doi: 10.1371/journal.ppat.1013263

Figure Lengend Snippet: (A) Schematic of virus neutralization by gH/gL-targeting nAbs. C57BL/6 mice were infected with 1,000 PFU MHV68 WT by intranasal inoculation. Serum was collected at 28 dpi (BioRender.com/6j9fwj6). (B) gH/gL-specific IgG from naïve or MHV68-infected C57BL/6 at a serum dilution of 1:80 was measured by MHV68 gL-gH ELISA. Background corrected optical density at 450 nm is shown. (C) Serum of MHV68-infected C57BL/6 mice neutralizes MHV68 infection on NIH 3T3 cells. MHV68 ORF59-GFP was pre-incubated with mouse serum at the indicated dilution. GFP expression was measured by flow cytometry. Infection is indicated as percentage of GFP + cells normalized to naïve serum. (D-E) Serum neutralization of MHV68 infection on NIH 3T3 cells is mediated by gH/gL-targeting antibodies. Antibodies to gH ecto /gL or gH D-I /gL were depleted using soluble complexes pre-coupled to magnetic beads. Fc was used as control. Neutralization was analyzed as in (C) at a serum dilution of 1:80. Micrographs were taken at 16 hpi. Mean and symbols representing individual experiments are shown. Statistical significance was evaluated by ordinary two-way ANOVA corrected for multiple comparisons by Tukey’s multiple comparisons test. ***: p-value < 0.001, ****: p-value < 0.0001.

Article Snippet: NIH 3T3 (RRID:CVCL_0594) and NIH 3T12 (ATCC CCL-164, Manassas, VA) murine fibroblasts were maintained in DMEM supplemented with 8% FCS, 100 IU/mL penicillin, 100 μg/mL streptomycin, and 2 mM L-glutamine (D8).

Techniques: Virus, Neutralization, Infection, Enzyme-linked Immunosorbent Assay, Incubation, Expressing, Flow Cytometry, Magnetic Beads, Control