Low-energy laser irradiation promotes the survival and cell cycle entry of skeletal muscle satellite cells
Gavriella Shefer1,
Terry A. Partridge2,
Louise Heslop2,
Jacqueline G. Gross2,
Uri Oron3 and
Orna Halevy1,*
1 Department of Animal Sciences, The Hebrew University of Jerusalem, P.O. Box
12, Rehovot 76100, Israel
2 Muscle Cell Biology Group, MRC Clinical Sciences Centre, Imperial College
School of Medicine, Hammersmith Hospital, Du Cane Road, London W12 0NN,
UK
3 Department of Zoology, The George S. Wise Faculty of Life Sciences, Tel Aviv
University, Tel Aviv 69978, Israel
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Fig. 1. LELI promotes cell cycle entry of cells attached to single fibers. BrdU was
added to control non-irradiated (A) or LELI (B) fibers immediately after
irradiation for 1 day, after which they were fixed and examined for BrdU
incorporation by immunohistochemistry. Most of the cells in the non-irradiated
fibers were negative for BrdU, whereas some were positive (brown) in the LELI
fibers. Arrows indicate some of the BrdU-positive cells. Bar, 30 µM.
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Fig. 2. LELI enhances the number of satellite cells emanating from single fibers.
Single myofibers prepared from EDL muscle of 21-day-old mice were
non-irradiated (left panels) or irradiated with He-Ne laser (right panels) and
kept in serum-free medium for the entire experiment. Data are expressed as the
cumulative rank of data from single fibers of four independent experiments
(n=200). Results are ranked according to the number of cells that
accumulated around each single fiber on the indicated days. Individual ranks
are expressed as percentage of total rank to normalize the data for sample
size, plotted on the vertical axis. Superimposed on each curve as an H symbol
is the mean value±1 s.e.m. for each treatment. Median value is
indicated by dotted lines. In most cases, the mean and median values are
close, therefore most samples are not distant from a Gaussian distribution.
The population distribution of LELI cells is significantly different from
control cells on all days (Kruskal-Wallis ANOVA median test; Rao R
(4,99)=9.65; P<0.001).
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Fig. 3. Analysis of the number of satellite cells emanating from single fibers in
the presence of serum. (A) Data are expressed as the cumulative rank of the
data from single fibers grown in DMEM containing 0.1% HS and either
non-irradiated (left panel) or LELI (right panel) (n=200). Curves
were plotted as described in Fig.
2. The population distribution of LELI cells is significantly
different from control cells on all days (Kruskal-Wallis ANOVA median test;
Rao R (4,99)=9.65; P<0.001). (B) A synergistic effect of LELI and
serum on cell accumulation around the fibers. Data are presented as the
percentage change of the total cell number that was pooled from all
experiments in each treatment relative to day 1. White bars, non-irradiated
fibers were grown in 0.1% HS-DMEM. Black bars, fibers were irradiated and kept
in serum-free DMEM. Hatched bars, fibers were subjected to LELI and kept in
DMEM-0.1% HS.
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Fig. 5. Survival of irradiated or non-irradiated i28 cells as measured by MTT assay
of mitochondrial function (A). (B) Hoechst staining for DNA nuclei, 24 hours
post irradiation. Note the fragmented/apoptotic nuclei versus the intact ones
in control and laser-treated cells, respectively. (C) Apoptotic nuclei were
counted and data are presented as a percentage of total nuclei. Each graph
represents mean±s.e.m of three replicates in which more than 1,000
cells were individually examined.
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© The Company of Biologists Ltd 2002
-tubulin. (F) Densitometric analysis of
protein expression levels normalized to that of