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. Author manuscript; available in PMC: 2015 Apr 1.
Published in final edited form as: Nat Protoc. 2014 Sep 18;9(10):2438–2449. doi: 10.1038/nprot.2014.168

Figure 6.

Figure 6

Analysis of CDH23 functions in OHCs. (a–d) OHCs are co-injectoporated at P1 with a plasmid expressing EGFP and an shRNA targeting CDH23 (shCDH23). Knockdown of CDH23 in EGFP-expressing cells is determined after 3 d in vitro (DIV) by immunohistochemistry. CDH23 (red) expression is no longer detectable in the stereocilia (white; Phal, phalloidin) of EGFP (green)-expressing OHCs. (e) Current displacement plots showing that mechanotransduction currents (METCs) are drastically reduced in OHCs expressing shCDH23 but not a scrambled control shRNA (shControl). The METC values (in pA) of controls are 75.98 ± 9.35 at 0.2 µm, 266.32 ± 26.86 at 0.4 µm, 362.50 ± 32.32 at 0.6 µm, 409.84 ± 36.37 at 0.8 µm and 439.23 ± 33.89 at 1.0 µm, respectively. In shCDH23-treated cells, 12.71 ± 3.33 at 0.2 µm, 35.00 ± 16.79 at 0.4 µm, 49.85 ± 27.04 at 0.6 µm, 61.87 ± 31.27 at 0.8 µm and 62.43 ± 36.50 at 1.0 µm. (f) Quantitative analysis of GCaMP3 signals in OHCs that were injectoporated at P1 to express shCDH23 or scrambled control shRNA. The tissues are cultured for 3 DIV and the OHCs are stimulated with a fluid jet with the same protocol as in Figure 5b. The values of ΔF/F0 are 0.93 ± 0.24 for shControl and 0.23 ± 0.07 for shCDH23. (g,h) OHCs from CDH23v2J/v2J mice are co-injectoporated at P1 with expression vectors for GCaMP3 and full-length CDH23 and analyzed after 4 DIV. (g) Cochlear whole-mounts are stained with Phal (red) to reveal stereocilia. GCaMP3 (blue) and CDH23 (green) are visualized with specific antibodies. Note that hair bundles in noninjectoporated cells of CDH23v2J/v2J mice are splayed (arrows). Overexpression of CDH23 (asterisks) rescues the morphological defect. (h) Mechanotransduction responses as analyzed by imaging GCaMP3 are perturbed in CDH23v2J/v2J mice but rescued by overexpression of CDH23. The values of ΔF/F0 are 0.87 ± 0.17 for CDH23+/v2J OHCs, 0.03 ± 0.01 for CDH23v2J/v2J OHCs and 0.72 ± 0.11 for CDH23v2J/v2J OHCs expressing CDH23. The number of analyzed hair cells is indicated. Values are mean ± s.e.m. Scale bars, 10 µm (a–d,g). ***P < 0.001. NS, not significant.