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. 2000 Aug 7;150(3):553–566. doi: 10.1083/jcb.150.3.553

Figure 5.

Figure 5

Day 4 MYC/M171-M183–transfected culture, triple stained with (A) anti-MYC, (A′) anti–s-α-actinin, and (A′′) DAPI. Asterisks mark several untransfected myotubes. Fine, diffuse MYC-positive granules are distributed throughout the transfected myotube. Their presence has not detectably interfered with the assembly of morphologically normal periodic Z-bands (A′). Arrows point to lateral sarcoplasmic extensions that are not occupied by SMFs. (B–B′′) Day 6 MYC/M171-M183–transfected culture, triple stained as above. In addition to the ectopic MYC-positive granules, note the ectopic slender nonstriated longitudinal filaments (arrows) that insert between normal SMFs (see below). Neither ectopic MYC-positive structure binds anti–s-α-actinin, nor does it act as a dominant negative. Occasionally, just perceptible MYC/Z-bands can be observed. As discussed in Materials and Methods, these MYC/Z-bands are not likely to be due to bleedthrough. Asterisks mark untransfected myotube. Day 6 MYC/M171-M183–transfected culture, triple stained with (C) anti-MYC, (C′) antinebulin (NB2), and (C′′) DAPI. Antinebulin (NB2) localizes to the endogenous nebulin. Note that ∼70% of the ectopic MYC-positive filaments/patches costain with antinebulin (NB2). Anti-MYC, but not antinebulin (NB2), accumulates along the edge of the lateral sarcoplasmic extensions (arrows), which lack all myofibrillar structures. MYC/Z-bands are more evident in this day 6 myotube than in younger myotube in Fig. 5 A. Asterisks mark two out-of-focus, overlapping untransfected myotubes. Day 10 MYC/M171-M183–transfected culture, triple stained with (D) anti-MYC, (D′) anti–s-α-actinin, and (D′′) DAPI. Note the elimination of the ectopic MYC-positive granules and filaments/patches in these older myotubes. Asterisks mark out-of-focus untransfected myotubes. Arrow points to an out-of-focus binucleated myotube. Day 4 myotubes expressing MYC/M175-M184. Triple stained with (E) anti-MYC (E′) anti-MHC, and (E′′) DAPI. Ectopic MYC-positive granules and fibrils/patches dominated these transfected cells. MYC-positive Z-bands were not evident. Nevertheless, the morphology of the 1.6-μm A-bands (E′) was normal. Asterisks mark untransfected myotube. Bar, 10 μm.