Expression of Myogenic Markers Is Not Affected after EB3 Depletion
(A) Western-blot analysis of 20,000 GFP-expressing cells after fluorescence-activated cell sorting (FACS), 3 days after transfection with shRNA constructs. Blots were probed with antibodies against EB1, EB3, α-tubulin, myogenin, Glu-tubulin, and embryonic myosin. Positions of molecular weight markers are indicated on the left.
(B) Immunofluorescence staining for embryonic myosin in EB3-depletion experiments after 55 hr in differentiation conditions. In the top panel, GFP-tubulin (white) highlights a transfected cell; in the bottom panel, embryonic myosin staining (white) of the same group of cells is shown. DNA is shown in blue. Scale bars represent 20 μm.
(C) Immunolocalization of PCM-1 (green) after 55 hr in differentiation conditions. GFP-tubulin (red) highlights transfected cells with either control (upper row) or EB3 shRNAs (lower row). DNA is shown in blue. Scale bars represent 20 μm.

Microtubule Behavior at the Cell Cortex is Altered in EB3-Depleted Cells
(A) Microtubule organization in C2C12 cells 3 days after transfection with shRNA constructs and after 52 hr under differentiation conditions. GFP-tubulin highlights the microtubule cytoskeleton in transfected cells. The insets show the cell edges of differentiating myoblasts after 49 hr under differentiation conditions. Scale bars represent 20 μm, and 2 μm in the insets.
(B) Microtubule organization in C2C12 cells 3 days after cotransfection of the EB3 shRNA construct and rescue constructs for full-length EB3 or EB1. GFP-tubulin highlights the microtubule cytoskeleton in transfected cells. Scale bars represent 20 μm.
(C) Images taken from time-lapse movies of GFP-tubulin in shRNA transfected cells that were shifted to differentiation for 49 hr. The time in seconds is indicated in the upper-right corner. Arrows highlight newly appearing bumps in microtubules that reach the cell cortex and continue growth. Asterisks indicate the relaxation of such bumps. The scale bar represents 2 μm. See the Supplemental Data for the respective movies.
(D) Quantification of the time microtubule tips spent at or close to the cortex (maximum of 2 μm away). Columns represent data obtained from 93–129 microtubules in five to eight cells, and error bars indicate SD. Microtubule tips spend significantly less time close to the cortex in EB3-depleted compared to control cells (analysis of variance [ANOVA]: p = 0.0003).
(E) Quantification of the time microtubules continue to grow after reaching the cell cortex, resulting in the introduction of bends into microtubules. Columns represent data obtained from 38–64 microtubules in four to eight cells, and error bars indicate SD.

EB Proteins during Myogenic Differentiation
(A) Morphological changes and reorganization of the microtubule cytoskeleton. Immunofluorescence of C2C12 cells that were shifted to differentiation for the times indicated and stained for α-tubulin (red), PCM-1 (green), and DAPI (blue) is shown. The scale bar represents 20 μm.
(B) Reverse-transcription polymerase chain reaction (RT-PCR) of EB1, EB2, EB3, and α-tubulin from RNA isolated from C2C12 myoblasts that were cultured under differentiation conditions for the times indicated.
(C) Western blots of total cell lysates of C2C12 cells that were cultured under differentiation conditions for the times indicated and probed with antibodies against EB1, EB2, EB3, and α-tubulin.
(D) C2C12 myoblasts, cultured under differentiation conditions for 41 hr, 3 days after transfection with shRNA constructs against luciferase (control), EB3, and EB1, coexpressing GFP-tubulin. The scale bar represents 50 μm.
(E) Average of the mean cell length in four to seven cell-elongation experiments after 19–54 hr under differentiation conditions. The average cell size of undifferentiated myoblasts is included for comparison. Error bars represent the standard deviation (SD).
(F) Myoblast fusion after 54 hr differentiation. GFP-tubulin (white) highlights cells that were transfected with the shRNA constructs (control and EB3); DNA staining is shown in blue. Clusters of nuclei (arrows) indicate myoblast fusion in both fields of view. Note that cells transfected with EB3 shRNAs do not participate in fusion. The scale bar represents 100 μm.
(G) Quantification of myoblast-fusion efficiency after 49–54 hr under differentiation conditions. Because of variations between experiments, the efficiency of cell fusion (percentage of cells containing two or more nuclei) was set to 100% for control cells in each experiment. Columns represent the average of three to four experiments, and error bars represent the SD.

Rescue of EB3 Depletion
(A) Quantification of myoblast fusion efficiency after 54–55 hr under differentiation conditions. The efficiency of cell fusion (percentage cells containing two or more nuclei) was set to 1.0 for control cells in each experiment (ranging from 8% to 34% fused cells), and relative values are shown for all other constructs. Columns represent an average of three to four experiments, and error bars indicate the SD. The columns representing values not significantly different from those of control cells (ANOVA: p > 0.05) are shown in light blue, whereas values not significantly different from those of EB3 deletion are shown in orange (ANOVA: p > 0.05). A partial rescue (which falls in neither of these categories, with p < 0.02) is shown in yellow.
(B) Western blots of extracts of GFP-positive cells (after FACS sorting) expressing the rescue constructs indicated in Figure S5, as well as EB3 shRNAs. Blots were probed with antibodies against FLAG and α-tubulin.
(C) Sequence alignment showing the first 53 amino acids of murine EB1 and EB3 (Swiss-Prot accession numbers Q61166 and Q6PER3, respectively). Identical amino acids are highlighted in blue, similar amino acids in orange, and sequence differences in yellow and red.
(D) Representation of the amino acid changes that confer partial rescue to the EB1 construct in the 3D structure of EB3 N-terminal domain (PDB accession number 1WYO, model 1, amino acids 8–137). The putative microtubule-binding site around K89 is shown in green [22], and the five crucial amino acid changes between EB1 and EB3 are shown in magenta or dark red. The image was created with Protein Explorer 2.78 (http://www.proteinexplorer.org).
(E) Surface maps of N-terminal domains (amino acids 1–130) of EB1 and EB3 were created with SYBYL 6.9.1 on the basis of PDB files 1PA7 and 1WYO. The four surface-exposed amino acid changes are highlighted in pink and red. Note that the surface structure changes as Y32 in EB3 fills a deep cleft present in EB1, and amino acids H29 and D26 emanate from the surface of the molecule.