JCS039255 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Specificity of EB1 targeting shRNAs. EB3 level and microtubule plus-end localization is not affected in EB1-knockdown NIH3T3 fibroblasts. EB1 was knocked down permanently in NIH3T3 cells with sh2 and sh3 RNAs and the cultures were stained for EB1 and EB3. Pooled transfected cells show different levels of residual EB1. Confocal images show that in cells highly depleted of EB1 (green, position marked with asterisks), the level and localization of EB3 (red) is not affected. Scale bar: 10 µm.

• Supplemental Figure S2 -

  Fig. S2. Overexpression of truncated EB1C-GFP but not of full-length EB1-GFP inhibits C2 myoblast fusion and myotube elongation. Widefield images of C2 cells expressing either EB1-GFP or EB1C-GFP after 2 days in FM. Fusion is reduced in EB1C-GFP-expressing cells (left bar graph), of which 64% are mononucleated and 36% multinucleated (n=591), compared with 27% mononucleated and 73% multinucleated in EB1-GFP-expressing cells (n=376). In addition, the cell shape of multinucleated myotubes differs depending on the construct expressed (right bar graph). Most EB1-GFP myotubes (93%, n=274) look normally elongated. By contrast, very few EB1C-GFP myotubes (9%, n=212) appear elongated; instead they are short and wide. For the calculations shown in the bar graphs, a myotube is considered as one cell regardless of the number of nuclei. Scale bars: 20 µm.

• Supplemental Figure S3 -

  Fig. S3. Glu microtubules are resistant to either cold or nocodazole treatment. Immunofluorescence of α-tubulin and glu-tubulin is shown in control C2 and in C2 cells treated with nocodazole (0.5 µg/ml for 1 hour at 37°C) or cold (30 minutes on ice). Cells were cultured in FM for 1 day, treated, and extracted with PHEM-TX-100 before fixation. Images shown are confocal z-stack projections taken under similar imaging parameters for each antibody. In control cells, both α-tubulin and glu-tubulin are detected in all cells and glu-tubulin is more abundant in myotubes than in the neighboring unfused cells. In nocodazole-treated cells, the remaining microtubules are glu-tubulin positive. In cold-treated cells, the remaining microtubules are labeled with glu-tubulin or are curly, suggesting that they are acetylated. Scale bar: 25 µm.

• Supplemental Figure S4 -

  Fig. S4. Cadherin and/or β-catenin localization in EB1-knockdown C2 and NIH3T3 cultures. (A) Immunofluorescence of EB1 and cadherin is shown in control C2 (top row) and C2-sh2F cells (bottom row) after 1 day in FM. Notice the weak cadherin staining in the cytoplasm and at the cell surface in an undifferentiated control myoblast (asterisks). Much stronger cadherin staining was found at plasma membrane of elongated, spindle-shaped control cells (arrows), which are known to be differentiated. The cadherin staining pattern observed in NIH3T3 fibroblasts (B) resembles that in the C2-sh2F, in which cadherin is found with β-catenin at cell-cell contacts (arrowheads), but not on the whole cell surface. Imaging conditions were optimized for each staining to highlight staining patterns. Scale bars: 10 µm.

• Supplemental Figure S5 -

  Fig. S5. EB1-GFP decorates microtubules in myoblasts and myotubes. Confocal images of C2 cultures transfected with EB1-GFP (green), fixed and stained with anti-glu-tubulin (red) 24 hours after transfection (myoblasts, A) or after 2 days in FM (B-C). In myoblasts and in myotubes with moderate expression levels, EB1-GFP decorates microtubule tips prominently; in myoblasts it also labels the centrosome (arrow in A). In myotubes with higher expression levels, labeling is observed all along the microtubules (C) (EB1-GFP, green; glu-tubulin, red; nuclei, blue). Scale bars (A): 20 µm; (B-C): 50 µm.