(A and B) Assessment of MET blockage with MET channel-permeable dye, FM1-43. (A) Left panels show maximal projection images of FM1-43 fluorescence in mouse organ of Corti explants immediately after the tissue dissection, in control conditions (top) and in the presence of non-saturating concentrations of MET blockers: amiloride (100 μM, middle) or benzamil (30 μM, bottom). Right panels show reference bright-field images of the same cochlear explants at the focal plane of the hair cell bodies. Data are representative of two independent series. (B) Similar maximal projection FM1-43 (left) and bright-field (right) images at the end of 24 hr incubation at 37°C with 30 μM of benzamil (top) and after washout of this drug (bottom). (C) Representative scanning electron microscopy (SEM) images of OHC stereocilia bundles (false-colored) in mouse organ of Corti explants cultured for 24 hr at 37°C in vehicle control conditions (top), 100 μM of amiloride (middle), or 30 μM of benzamil (bottom). Right panels show higher magnification images of OHC stereocilia. Arrows point to examples of retracted stereocilia. (D) Heights of individual stereocilia in different rows of OHC bundles in mouse organ of Corti explants cultured for 24 hr in control conditions (black; n = 103–120 stereocilia) or in the presence of the MET blockers (gray), amiloride (100 μM, n = 99–108) or benzamil (30 μM, n = 75–80). Error bars indicate mean ± SD. The data are from a single series of experiments (8–17 cells per treatment) with control and drug-treated explants processed in parallel, representative of one (amiloride) and three (benzamil) independent series. (E and F) Representative false-colored SEM images of OHC bundles (E) and quantification of stereocilia heights (F) in the first (blue), second (yellow) and third (red) rows of the bundle (n = 40–130), indicating the dose-dependent effect of a 32 hr incubation in the presence of 0, 5.5, 10 and 30 μM of benzamil. Data (4–12 cells per treatment) are shown as mean ± SD. For D and F: *p<0.05; ****p<0.0001; n.s., non-significant (Welch’s t tests). (G) Percentage of shorter (second and third) row OHC stereocilia having tip links after 24 hr culturing in control conditions (black, n = 632) or with MET blockers (gray, n = 262–527). Combined data from two independent series (7–17 cells per treatment) are shown as mean ± SE. n.s., non-significant (Student’s t tests). Age of explants A: P5; B–G: P4 +24–32 hr incubation. Original SEM images can be found in Vélez-Ortega et al. (2017).