FIGURE 4

FIGURE 4

Impact of azithromycin and neomycin treatment on hair cell function. (A) AZM (190 µM) does not significantly hinder FM1-43 uptake into lateral line HCs (p = 0.15), as quantified by PEPITA. This suggests that AZM does not inhibit MET function. In contrast, administration of the MET inhibitor benzamil (BZM; 50 µM) as a positive control does significantly reduce FM1-43 uptake (p < 0.0001). (B) Effect of AZM (190 µM) co-administration on accumulation of TexasRed-conjugated neomycin (NEO-TR, 50 μM exposure) within myo6b::gfp lateral line HCs, as quantified by PEPITA. BZM (50 µM) co-administration with NEO was also evaluated as a positive control, and both conferred inhibition of NEO-TR uptake into the hair cells (BZM, p = 0.001; AZM, p = 0.0001). (D and E) Representative fluorescence microscopy images of the colocalization of NEO-TR (TexasRed (TxRed), red) with GFP-expressing HCs (GFP, green), treated with NEO-TR alone (D) or with both NEO-TR and AZM (E) for 30 min, after washout, not used for quantification of NEO-TR accumulation or GFP fluorescence levels. (C) Quantification of mitochondrial Ca²⁺ levels in individual HCs in response to NEO treatment alone (blue) or NEO plus AZM co-administration (orange), as measured by mitoGCaMP3 fluorescence signal. Drugs were administered at t = 10 min; (F and G) Fluorescence microscopy images of mitoGCaMP3 neuromasts at 30 min post drug administration, treated with NEO alone (F) or with both NEO and AZM (G). Scale bar for panels (D–G) represents 20 µm. The dashed shapes in panels F and G depict example regions of interest (ROI) used to quantify fluorescence signal.