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First published online 28 October 2008
doi: 10.1242/jcs.024547

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A non-apoptotic role for caspase-9 in muscle differentiation

National Centre for Biomedical Engineering Science and Department of Pharmacology and Therapeutics, National University of Ireland, Galway, Ireland

View larger version (43K): [in this window] [in a new window]   Fig. 1. Non-apoptotic morphology of adherent C2C12 cells and caspase-3 activity in the adherent cells. C2C12 cells were induced to differentiate over a 5-day time course. (A) DAPI-stained nuclear morphology of adherent and detached C2C12 cells after 2 days of differentiation. (B) Caspase-3-like activity was assayed using the fluorogenic substrate DEVD-afc over a 5-day differentiation period. Enzyme activity is expressed as an increase in arbitrary fluorescent units per minute per milligram of protein (AFU/min/mg). The histogram shows the mean ± s.d. of three independent experiments. (C) Immunoblot for caspase-3 processing over a 5-day differentiation period. Staurosporine-treated (1 µM for 4 hours) C2C12 cells (+ve) were used as a positive control for caspase-3 cleavage.

View larger version (42K): [in this window] [in a new window]   Fig. 2. A caspase inhibitor impairs differentiation. A broad-spectrum pharmacological caspase inhibitor, Q-VD-OPH, was used at 30 µM to inhibit caspase activity in differentiating C2C12 cells. Cells were incubated in differentiation media for the indicated time, and caspase-activity assays were conducted and MyHC expression analysed. The histograms show the mean ± s.d. of three independent experiments. (A) Caspase-3-like activity in differentiating C2C12 cells in the presence (grey) or absence (black) of 30 mM Q-VD-OPH. Enzyme activity is expressed as an increase in arbitrary fluorescent units per minute per milligram of protein (AFU/min/mg). (B) MyHC (green) expression during differentiation with DNA counterstaining with Hoechst 33342 (blue). (C) The degree of muscle-cell differentiation was determined by scoring cell fusion (see Materials and Methods). (D) The number of nuclei per field was determined for each treatment.

View larger version (59K): [in this window] [in a new window]   Fig. 3. Caspase-9 is activated during differentiation. (A) Caspase-9 processing was detected by immunoblotting over a 5-day differentiation time course. Staurosporine-treated (1 µM for 4 hours) C2C12 cells (+ve) were used as a positive control for caspase cleavage. (B) Affinity labelling of the 37-kDa band of processed caspsase-9. C2C12 cells were cultured for 12 hours in growth media (GM) or differentiation media (DM) in the presence or absence of 50 µM biotin-VAD-fmk. Labelled caspases were affinity purified and detected by immunoblot.

View larger version (28K): [in this window] [in a new window]   Fig. 4. shRNA against caspase-9 impairs muscle differentiation. C2C12 cells were transduced with either an adenoviral vector encoding shRNA targeted against caspase-9 (AdshRNA-C9) or the same vector without the shRNA (AdNull). Caspase-9 levels were assessed by immunoblot (A) and the effect of shRNA on caspase-3-like activity was assessed by DEVD-afc (B). Enzyme activity is expressed as an increase in arbitrary fluorescent units per minute per milligram of protein (AFU/min/mg). (C) The effect of AdshRNA-C9 knockdown on caspase processing was determined by immunoblot. Arrows indicate the processed forms of caspase-9 and caspase-3. (D) The effect of AdshRNA-C9 knockdown on cell fusion (see Materials and Methods) was assessed. The histograms show the mean ± s.d. of three separate experiments. (E) Top, western blot for caspase-9 in stained samples. Bottom, MyHC (green) staining with DNA (blue) counterstaining (Hoechst 33342).

View larger version (28K): [in this window] [in a new window]   Fig. 5. Bcl-xL overexpression impairs differentiation. C2C12 cells were transduced with either an adenoviral vector encoding the anti-apoptotic protein Bcl-xL (AdBcl-xL) or with the same vector without the transgene (AdNull). Bcl-xL expression was assessed by immunoblot (A) and caspase-3-like activity was assayed using DEVD-afc (B). Enzyme activity is expressed as an increase in arbitrary fluorescent units per minute per milligram of protein (AFU/min/mg). The effect of Bcl-xL expression on caspase processing (C) and on MyHC expression (D) was assessed by immunoblot and immunohistochemistry. MyHC is shown in green whereas DNA counterstaining (Hoechst 33342) is shown in blue. (E) The degree of muscle-cell differentiation was determined as before (see Materials and Methods). The histograms show mean + s.d. of three separate experiments.

View larger version (21K): [in this window] [in a new window]   Fig. 6. Number of MyHC-expressing cells per field. C2C12 cells were induced to differentiate after the indicated treatments, then harvested and fixed in graded methanol. Fixed cells were immunostained for MyHC expression. The number of nuclei per MyHC-positive cell (A) and the number of MyHC-positive cells per microscopical field (B) were scored. The histograms show the mean ± s.d. of three independent experiments. *P=0.005.

View larger version (22K): [in this window] [in a new window]   Fig. 7. Mitochondrial changes in apoptotic but not differentiating cells. C2C12 cells were treated with staurosporine (+ve) to induce apoptosis or were induced to differentiate. Differentiating cultures were washed to remove non-adherent cells. Cells were then harvested, lysed and centrifuged to obtain cytosolic and mitochondrial subcellular fractions. (A) The presence of mitochondrial proteins in the cytosolic fractions was then detected by immunoblotting. (B) Mitochondrial contamination of subcellular fractions was assessed by immunoblotting for COX IV. The results shown are typical of three independent experiments. (C,D) Alternatively, cells were harvested and stained with TMRE to detect m by flow cytometry. The results shown are typical of three independent experiments.

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