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First published online 31 July 2007
doi: 10.1242/jcs.004002

Journal of Cell Science 120, 2904-2911 (2007)
Published by The Company of Biologists 2007
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Brg1 chromatin remodeling factor is involved in cell growth arrest, apoptosis and senescence of rat mesenchymal stem cells

Marco A. Napolitano1, Marilena Cipollaro1, Antonino Cascino1, Mariarosa A. B. Melone2, Antonio Giordano3 and Umberto Galderisi1,3,*

1 Department of Experimental Medicine, Section of Biotechnology and Molecular Biology, Excellence Research Center for Cardiovascular Diseases, Second University of Naples, Naples, Italy
2 Department of Neurology, Second University of Naples, Naples, Italy
3 Sbarro Institute for Cancer Research and Molecular Medicine, Center for Biotechnology, Temple University, Philadelphia, PA, USA


Figure 1
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  		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.

 

Figure 2
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  		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.

 

Figure 3
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  		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 beta-tubulin, chosen as an internal control. Each experiment was repeated at least three times. Variations in protein expression are given as arbitrary units.

 

Figure 4
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  		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.

 

Figure 5
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  		Fig. 5. Senescence-associated beta-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 {perp}) and Rb (Rb {perp}), respectively. The picture shows several senescent cells as detected with beta-galactosidase assay. White asterisk, a typical flat large senescent cell.

 

Figure 6
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  		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.

 

Figure 7
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  		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).

 

Figure 8
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  		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 2x Laemmli sample buffer and western blots were performed to detect Brg1. Lanes 1 and 4, input protein lysates.

 

Figure 9
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  		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).

 

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© The Company of Biologists Ltd 2007