DEV030718 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Specificity of antibodies against MYH9, MYH10 and MYH14. Cochlear tissue was dissected at E16.5 as described in the Materials and methods section. Total protein was extracted by incubation of cochlear epithelia or adult brain tissue in RIPA buffer (Sigma) with NaCl (added to a final concentration of 300 µM), Protease Inhibitor Cocktail (Complete Mini, Roche) and Phosphatase Inhibitor Cocktail (Sigma). The lysates were centrifuged at 10,000 g for 15 minutes at 4°C, and the supernatant was denatured by incubation at 70°C for 10 minutes with NuPAGE LDS Sample Buffer (Invitrogen) and β-mercaptoethanol (Sigma). The denatured samples were fractionated by SDS-PAGE on a 4-12% gradient Bis-Tris gel (Invitrogen) and transferred onto nitrocellulose membranes (Invitrogen). Proteins were visualized using the Amersham ECL plus Western Blotting Detection System (GE Healthcare) according to the manufacturer's instructions. Primary antibody dilutions were as follows: mouse monoclonal anti-β-actin (Sigma-Aldrich, 1:50,000), rabbit polyclonal anti-MYH9 (Covance, 1:500), rabbit polyclonal anti-MYH10 (Covance, 1:500) and rabbit polyclonal anti-MYH14 (1:1000). Specific bands at the appropriate predicted molecular weights were obtained for each antibody. No additional bands were observed in any sample.

• Supplemental Figure S2 -

  Fig. S2. Expression of Myh genes in the developing cochlea and other tissues. (A) Results from RT-PCRs for each of the MYH genes from different adult tissues and embryonic cochleae. Arrowheads indicate the predominant bands for each transcript. Arrows indicate known splice variants for each gene. Multiple primer sets were used for Myh10 to detect different Myh10 isoforms. However, none of the variants was detected in cochleae at either age. In contrast, all isoforms of Myh14 were detected in cochlear tissue. (B-D) Quantitative RT-PCR for changes in relative expression of each MYH gene in cochlear tissue between E12.5 and E16.5. Myh9 and Myh10 levels drop to between 50% and 60% of maximal values between E12.5 and E16.5, whereas the level of Myh14 expression increases nearly sixfold during the same time period.

• Supplemental Figure S3 -

  Fig. S3. Distribution of MYH9 and no primary control. (A) Distribution of anti-MYH9 in the middle and basal regions of the cochlea at E16.5, labeled as indicated. In the middle region, MYH9 distribution is weak and diffuse. In the basal region, MYH9 labeling is still present with a possible increase in the inner hair cell region. (B) No primary control in an E16.5 cochlea labeled as in A. No non-specific staining was observed. Scale bar: 25 µm (same magnification in B).

• Supplemental Figure S4 -

  Fig. S4. Cross-sections through the basal regions of wild-type (Myh10^+/+;Foxg1^Cre/+) and Myh10 dominant negative (Myh10^DN/DN;Foxg1^Cre/+) cochleae at E16. The thickness of the epithelium along the basolumenal axis is similar in both cochleae. Sections in the upper panels are labeled with phalloidin to illustrate cell-cell boundaries. In the lower panels, sections are double labeled with DAPI (blue), to illustrate cell nuclei, and anti-SOX2 (red), to define the sensory epithelium. Scale bar: 50 µm.