Journal: The Journal of Cell Biology

Article Title: A novel mechanism of myocyte degeneration involving the Ca 2+ -permeable growth factor–regulated channel

doi: 10.1083/jcb.200301101

Figure Lengend Snippet: Characterization of CHO cells expressing GRC. (a) Effect of FCS on GRC localization in CHO cells serum-starved for 24 h. Cells were stained with anti-GRC or rhodamine-phalloidin. (b) Gd 3+ -inhibitable 45 Ca 2+ uptake into GRC-transfected or nontransfected cells measured for 5 min with cyclic stretch applied for 3 min starting from the end of the first min. (c) Stretch–induced changes in [Ca 2+ ] i as monitored by the fluo-4 fluorescence. The maximal increments of fluorescence are shown in the bar graph (mean ± SD). Microscopic fields before and after stretch showing low and elevated levels of fluorescence in GRC-expressing cells (bottom). Data represent a typical result from four similar experiments. (d) Effect of uniaxial cyclic stretch on the cell orienting response. Rhodamine-phalloidin–stained images of nonstretched and stretched cells are shown. In b and c, error bars show means ± SD and asterisks show P < 0.05.

Article Snippet: In some experiments, we loaded cells with 2 μM fura-2 acetoxymethyl ester in place of fluo-4 acetoxymethyl ester and measured [Ca 2+ ] i by means of a ratiometric fluorescence method, using a fluorescence image processor (model Argus 50; Hamamatsu Photonics) mounted on an inverted microscope (40× objective; Nikon).

Techniques: Expressing, Staining, Transfection, Fluorescence

Journal: The Journal of Cell Biology

Article Title: A novel mechanism of myocyte degeneration involving the Ca 2+ -permeable growth factor–regulated channel

doi: 10.1083/jcb.200301101

Figure Lengend Snippet: Increased Ca 2 + influx into and CK efflux from BIO14.6 myotubes and their correction by δ-SG gene transfer. (a) 45 Ca 2+ uptake into normal or BIO14.6 myotubes measured under resting conditions with or without SK&F96365 (SK&F), ruthenium red (RR), or Nifedipine. The Gd 3+ -inhibitable fractions are shown. (b) External Ca 2+ -induced changes in fluo-4 fluorescence in normal or BIO14.6 myotubes. The bar graph shows the maximal increments of fluorescence in myotubes pretreated with or without 50 μM SK&F96365. ΔF/F 0 is the ratio between the fluorescence increment and the fluorescence before Ca 2+ addition. Numbers in parentheses correspond to the number of cells studied. (c) CK efflux from BIO14.6 myotubes subjected to cyclic stretch under indicated conditions. (d) Immunoblot assay and immunohistochemistry (IH) of BIO14.6 myotubes infected with Ad.β-gal or Ad.δ-SG. (e and f) External Ca 2+ -induced changes in fluo-4 fluorescence and cyclic stretch–induced CK efflux in BIO14.6 myotubes insfected with Ad.β-gal or Ad.δ-SG. TG, thapsigargin (1 μM). Other conditions were similar to b and c. In these panels, error bars show means ± SD and asterisks show P < 0.05.

Article Snippet: In some experiments, we loaded cells with 2 μM fura-2 acetoxymethyl ester in place of fluo-4 acetoxymethyl ester and measured [Ca 2+ ] i by means of a ratiometric fluorescence method, using a fluorescence image processor (model Argus 50; Hamamatsu Photonics) mounted on an inverted microscope (40× objective; Nikon).

Techniques: Fluorescence, Western Blot, Immunohistochemistry, Infection

Journal: The Journal of Cell Biology

Article Title: A novel mechanism of myocyte degeneration involving the Ca 2+ -permeable growth factor–regulated channel

doi: 10.1083/jcb.200301101

Figure Lengend Snippet: Effect of GRC antisense expression on BIO14.6 myotubes. (a) Immunoblot assay for GRC and β-dystroglycan (β-DG; top) and GRC immunohistochemistry (middle) of BIO14.6 myotubes infected with Ad.β-gal or Ad-antisense–GRC cDNA (Ad.asGRC). Cell surface GRC levels were estimated by labeling antisense-treated or nontreated myotubes with NHS-biotin and by further analyzing streptavidin agarose-bound (B) and -unbound (U) fractions by immunoblot assay with anti-GRC (bottom). (b and c) External Ca 2+ -induced changes in fluo-4 fluorescence and cyclic stretch-induced CK efflux from antisense-treated or nontreated myotubes. Other conditions were the same as those in (b and c). Error bars show means ± SD and asterisks show P < 0.05.

Article Snippet: In some experiments, we loaded cells with 2 μM fura-2 acetoxymethyl ester in place of fluo-4 acetoxymethyl ester and measured [Ca 2+ ] i by means of a ratiometric fluorescence method, using a fluorescence image processor (model Argus 50; Hamamatsu Photonics) mounted on an inverted microscope (40× objective; Nikon).

Techniques: Expressing, Western Blot, Immunohistochemistry, Infection, Labeling, Fluorescence