Article Figures & Tables
- Table 1.
Protein expression in MSCs transduced with Ad-CMV and Ad-CMV-Brg1
Ad-CMV-Brg1 Ad-CMV p53 0.51±0.02 0.26±0.03 pRb 0.4±0.02 0.12±0.03 pRb2/p130 0.21±0.05 0.11±0.02 p21 1.65±0.1 1.0±0.05 p27 0.08±0.01 0.16±0.02 - Fig. 1.
FACS analysis of MSCs transduced with Ad-CMV and Ad-CMV-Brg1. Analyses were performed 1 and 3 days after transduction. Pre G1 indicates sub-diploid cells that are undergoing programmed cell death.
- Fig. 2.
Semiquantitative RT-PCR analysis of mRNA expression of the indicated genes of MSCs transduced with Ad-CMV and Ad-CMV-Brg1. Analyses were performed 3 days after transduction. mRNA levels were normalized with respect to HPRT, chosen as an internal control. Each experiment was repeated at least three times.
- Fig. 3.
Western blot of protein expression in MSCs transduced with Ad-CMV and Ad-CMV-Brg1. Analyses were performed 3 days after transduction. Protein levels were normalized with respect to β-tubulin, chosen as an internal control. Each experiment was repeated at least three times. Variations in protein expression are given as arbitrary units.
- Fig. 4.
Semiquantitative RT-PCR analysis of mRNA expression of the indicated genes of MSCs transduced with Ad-CMV and Ad-CMV-Brg1. Analyses were performed 3 days after transduction. Each experiment was repeated at least three times.
- Fig. 5.
Senescence-associated β-galactosidase assay performed on MSCs transduced with the recombinant adenoviruses indicated in Table 2. Assays were performed 3 days after transductions. Ad-YH47 and Ad-RG2 indicate adenoviruses carrying mutated E1A isoforms that inhibit p53 (p53 ⊥) and Rb (Rb ⊥), respectively. The picture shows several senescent cells as detected with β-galactosidase assay. White asterisk, a typical flat large senescent cell.
- Fig. 6.
Polyacrylamide gel electrophoresis of TRAP assay products obtained from protein extracts of MSCs transduced with Ad-CMV and Ad-CMV-Brg1. Analyses were performed 3 days after transduction. The picture shows the PCR-amplified products of telomerase activity. Three different reactions for each transduction experiment are shown. Black arrow, TRAP internal control.
- Fig. 7.
Semiquantitative RT-PCR analysis of mRNA expression of the indicated genes of MSCs transduced with Ad-CMV and Ad-CMV-Brg1. Analyses were performed 3 days after transduction. mRNA levels were normalized with respect to HPRT, chosen as an internal control. Each experiment was repeated at least three times. The picture shows experiments performed on MSCs grown in proliferating medium (control medium) and adipocyte differentiation medium (inducing medium).
- Table 3.
TUNEL assay data
TUNEL(+) cells Ad-Brg1 11±1% Ad-Brg1+Ad-E1AYH47 6±0.9% Ad-Brg1+Ad-E1ARG2 12±1.1% Ad-CMV 1±0.8% - Fig. 8.
Immunoprecipitation experiments. Cells lysates were immunoprecipitated with anti-Rb (lane 2), anti-p53 (lane 5) and control IgG (lanes 3 and 6). The immunoprecipitates were resuspended in a 2× Laemmli sample buffer and western blots were performed to detect Brg1. Lanes 1 and 4, input protein lysates.
- Fig. 9.
Micrococcal nuclease assay. (A) Agarose gel electrophoresis of genomic DNA from MSCs transduced with Ad-CMV and Ad-CMV-Brg1. DNA was digested with micrococcal nuclease for various times, as indicated. The picture shows a high molecular weight DNA band that is more resistant to nuclease digestion in Ad-Brg1 samples than in Ad-CMV samples (see arrow). (B) DNA resolved on agarose gel following nuclease digestion as in A. MSCs were transduced with Brg1 and with viruses carrying E1A, E1A-YH47 and E1A-RG2 isoforms. These inhibit both p53 and Rb (E1A) or p53 (E1A-YH47) or Rb (E1A-RG2) alone. The picture shows high molecular weight DNA bands and faint smears of digested DNA molecules (see arrows).
Article navigation
Related articles
Cited by...
More in this TOC section
Similar articles
Other journals from The Company of Biologists
Editorial – “What I cannot create, I do not understand”
Journal of Cell Science is pleased to annouce an upcoming special issue on ‘Reconstituting cell biology’, guest edited by Manuel Théry (Hôpital St Louis, Paris and CEA Grenoble). In an introductory Editorial, Editor-in-Chief Michael Way reflects on current understanding of how the different components and systems of the cell are integrated and organized into higher form and function.
We invite you to submit your breakthrough research on all aspects of reconstituting cell biology for this special issue. The deadline for submissions is Feb 15th, 2018. Find out more here.
Cell scientist to watch – Paul Timpson
“[…] it's really positive and very important to show true translation of the work to the public.”
Paul Timpson leads a group at the Garvan Institute of Medical Research in Sydney, Australia, assessing cancer in the context of its surrounding environment with the use of advanced intravital imaging technology. In this interview, he shares why he feels it’s crucial to share his research in an accessible and accurate way.
Look out for First Person, our new interview series with early-career researchers. In this issue, we hear from Chaitali Khan, Meraj Hasan Khan and Seth Zimmerman.
Review – The constriction and scission machineries involved in mitochondrial fission
Mitochondria use dynamin and related proteins to constrict the outer membrane from the cytosolic face when dividing. In this Review, Felix Kraus and Michael T. Ryan summarise the role of proteins from the dynamin superfamily in mitochondrial division.
Transfer to Biology Open
If your submission to Journal of Cell Science is unsuccessful, did you know you can transfer your paper and any reviews directly to our sister journal, Biology Open? The majority of papers transferred with reviews are accepted for publication. Find out how here.