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First published online 9 March 2004
doi: 10.1242/jcs.01028

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Partitioning of IGFBP-5 actions in myogenesis: IGF-independent anti-apoptotic function

Signalling Programme, The Babraham Institute, Cambridge CB2 4AT, UK

View larger version (29K): [in a new window]   Fig. 1. Expression, IGF binding ability, and effects on Igf2 mRNA levels of wtIGFBP-5 and mutIGFBP-5. (A) Western immunoblot to detect the Myc epitope in cell lysates from C2 myoblasts transiently transfected with control vector, wtIGFBP-5 (wtBP5) or mutIGFBP-5 (mutBP5) and transferred to DM for 24, 48 and 72 hours. (B) Ligand blot using biotinylated IGF-I to detect IGFBP-5 in cell lysates from C2 myoblasts transfected as in A and harvested after 48 hours in DM. (C) Northern blot to detect Igf2 mRNA in myoblast lysates after transfection as in A and harvested after 72 hours; (upper panel) the major Igf2 transcript at 3.9 kb and (lower panel) ethidium bromide staining of the 28S and 18S RNA bands as a loading control.

View larger version (96K): [in a new window]   Fig. 2. WtIGFBP-5 but not mutIGFBP-5 inhibits myogenesis. (A) Haematoxylin and Eosin staining of C2 cells transfected with control vector, wtIGFBP-5 (wtBP5) or mutIGFBP-5 (mutBP5), 72 hours after transfer to DM (5x magnification). (B; upper panel) Fluorescent immunocytochemistry to show myosin heavy chain (MHC) protein in myoblasts transfected as in A; (lower panel) DAPI staining of nuclei (40x magnification).

View larger version (16K): [in a new window]   Fig. 3. WtIGFBP-5 and mutIGFBP-5 increase myoblast cell number but do not change proliferation rate. (A) Cell number, expressed as percentage of cells in GM compared with DM, in myoblasts transfected with control vector, wtIGFBP-5 (wtBP5) or mutIGFBP-5 (mutBP5); means±s.e.m.; n=3. (B) BrdU incorporation into myoblasts transfected as in A; mean±s.e.m., n=3.

View larger version (18K): [in a new window]   Fig. 4. WtIGFBP-5 and mutIGFBP-5 inhibit apoptosis in myoblasts. (A) Flow cytometric determination of the percentage of annexin V-positive, PI-negative myoblasts transfected with either control vector (C), wtIGFBP-5 (wtBP5) or mutIGFBP-5 (mutBP5) as an indication of apoptosis levels in C2 myoblasts after 24 hours in DM; mean±s.e.m., n=3; *P<0.05 compared with control vector; **P<0.01 compared with control vector. (B) Caspase-3 activity in myoblast lysates after transfection as in A, expressed as a fraction of control vector transfected cells at each time point after transfer to DM; means±s.e.m., n=3; significance that mean is less than 1.0: *P<0.05; **P<0.01.

View larger version (16K): [in a new window]   Fig. 5. Caspase-8 and caspase-9 activities in myoblasts during differentiation. Myoblasts were transfected with either control vector (C), wtIGFBP-5 (wtBP5) or mutIGFBP-5 (mutBP5) and (A) caspase-8, and (B) caspase-9 activities were determined in cell lysates during differentiation; means±s.e.m., n=3; data are expressed as a fraction of control values; significance that mean is less than 1.0: *P<0.05; **P<0.01.

View larger version (42K): [in a new window]   Fig. 6. WtIGFBP-5 and mutIGFBP-5 prevent the decrease in myoblast number induced by expression of antisense Igf2. Enriched myoblast populations expressing antisense Igf2 or vector (V) were additionally transfected with wtIGFBP-5 (wtBP5) or mutIGFBP-5 (mutBP5) or their control vector (C). Cell number is expressed as a percentage of values at the time of transfer from GM to DM for myoblasts at (A) 24 hours and (B) 48 and 72 hours in DM; means±s.e.m., n=3; **P<0.01 compared with V.

View larger version (28K): [in a new window]   Fig. 7. WtIGFBP-5 and mutIGFBP-5 ameliorates the increased apoptosis induced by antisense Igf2 expression in myoblasts. Enriched myoblast populations expressing antisense Igf2 or vector (V) were additionally transfected with wtIGFBP-5 (wtBP5) or mutIGFBP-5 (mutBP5) or their control vector (C). (A) Flow cytometric determination of the percentage of annexin V-positive, PI-negative myoblasts after 48 hours in DM. (B) Caspase-3 activity in lysates derived from myoblasts transfected as in A. For A and B, data are expressed as a fraction of the values obtained for myoblasts transfected with antisense Igf2 alone; means±s.e.m., n=3; significance that mean is less than 1.0: *P<0.05; **P<0.01. ***P<0.001.

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