Smc3-KD leads to decreased kinociliary axoneme length in the first neuromast along the posterior lateral line, which is rescued by smc3 mRNA overexpression. (A) Representative images of the first posterior lateral line kinocilium in SC-KD (left), Smc3-KD (right). Larvae were fixed at 3 dpf. Acetylated tubulin is labeled in red, and DAPI is in blue. Arrows identify the base and tip of each measured kinocilium. Scale bar: 10.4 µm in the x and y dimensions. (B) Quantification of the decrease in axoneme length in Smc3-KD (Student's t-test, ****P<0.0001, SC-KD n=35, Smc3-KD=26). (C) Representative images of the first posterior lateral line kinocilium in SC-KD+smc3 mRNA (left), and Smc3-KD+smc3 mRNA (right). Scale bar: 10.4 µm. (D) smc3 mRNA co-injection in Smc3-KD rescues axoneme length as compared Smc3-KD alone (Student's t-test, **P=0.0123, Smc3-KD+smc3 mRNA n=26, Smc3-KD n=26) smc3 mRNA co-injection in SC-KD decreases axoneme length as compared SC-KD alone (Student's t-test, *P=0.0228, SC-KD+smc3 mRNA n=19, SC-KD n=35).

Smc3 KD results in decreased hair cell number in neuromasts along the lateral line. (A) 3D maximum projection representative images of SC-KD (left) and Smc3-KD (right) MI1 neuromasts observed. Hair cells are visualized with an HCS-1 antibody at a 1:100 dilution to label Otoferlin. Scale bar: 8.4 µm in the x and y dimensions. (B) Quantification of the decrease in observed hair cells per neuromast in 5 dpf Smc3-KD larvae as compared to SC-KD. Student's t-test, P<0.0001. Five neuromasts were observed per embryo n=15, Smc3-KD embryo n=23.

Smc3 protein depletion protects lateral line neuromasts from neomycin cytotoxicity. (A) Representative images of lateral line neuromasts in SC-KD larvae and Smc3-KD larvae at 5 dpf with and without neomycin treatment and stained with 2-[4-(Dimethylamino)styryl]-1-ethylpyridinium iodide (DASPEI). Scale bars: 100 µm. (B) Quantification of neuromast survival after neomycin treatment for each injection type. Neuromast survival was calculated as the ratio of observed neuromasts in each neomycin-treated embryo to the average number of neuromasts visible in control-treated embryos of the same injection type (e.g. SC-KD or Smc3-KD). By this method, ratios greater than 1 may occur if the number of observed neuromasts in a neomycin-treated embryo exceeds the average number of neuromasts observed in the relevant control-treated embryos. Student's t-test, ****P<0.0001, SC-KD n=40, Smc3-KD n=36.

Smc3 protein depletion does not significantly impact FM1-43FX uptake via mechanotransduction. (A) Maximum projection representative images of first posterior lateral line neuromasts of each injection type following brief FM1-43FX treatment in 6 dpf embryos. (B) Quantification of the average fluorescence intensity normalized to background fluorescence of neuromasts of each injection type. Scale bar: 10.4 µm in the x and y dimensions. Student’s t-test, P=0.3646, SC-KD n=14, Smc3-KD n=11.