Supplementary MaterialsS1 Fig: Tmie-GFP shows variable expression in stereocilia. E is the whole hair cell, and in C and F is a subtraction of whole cell fluorescence minus soma fluorescence to roughly determine the relative contribution of bundle signal. Significance was determined by two-tailed unpaired t-test with Welchs correction, **p 0.01, ****p CSPG4 0.0001.(TIF) pgen.1007635.s002.tif (1.0M) GUID:?C7360E0A-46DD-4A5B-AA3C-A6AE3D647BC4 S3 Fig: Differential effects on function with a genomic mutation and a transgene mimic. (A) Data for a novel mutant allele of (below) showing the genomic region where the mutation occurs. An arginine is mutated to guanine in the splice acceptor (dark package, above) of the ultimate exon of larvae bridging exons 3 and 4. Proteins: The expected proteins products, shown right here like a two-pass transmembrane proteins. The crazy type proteins has many billed residues (positive in light grey, adverse in dark grey) that are dropped in larvae, used having a hand-held Cannon camera. Arrow factors to a larva that’s upside-down, displaying a vintage vestibular phenotype. (B) Top-down look at of a consultant neuromast after exposure to FM 4C64, imaged using confocal microscopy. The first panel is a single plane through the soma region while the second panel is a maximum projection of 7 panels through the soma region, beginning at the cuticular plate (as denoted by magenta bracket in Fig 1G). (C) Same as (B) except that the first panel shows the bundle region so that 1-138-GFP can be visualized in bundles (as depicted by dashed green line, Fig 1G). The transgene is driven by the promoter. (D) Plot of the integrated density of FM fluorescence per cell. We normalized values order Abiraterone to the average of wild type siblings. Displayed wild type and data are from siblings of and are the same values reported in Fig 6. Data for is from a separate experiment. Statistical significance determined by one-way ANOVA, ****p 0.0001. Scale bar is 10m.(TIF) pgen.1007635.s003.tif (3.5M) GUID:?7AAA631D-EEC2-4675-831E-68A6DE67C18C S4 Fig: Expression pattern and functional rescue by constructs CD8 and 139C231. All images were captured using confocal microscopy. (A) Stereocilia of a neuromast viewed from above. The same neuromast was imaged at 4 dpf and 6 dpf. In hair cells expressing CD8-GFP, signal was initially detected in immature bundles, but this expression was only detectable in soma by dpf 6 as the cells matured (n = 10 cells). (B) Maximum projection of neuromasts viewed from above; left panel shows only FM 4C64 while right panel adds CD8-GFP. No rescue of FM 4C64 order Abiraterone labeling was observed in hair cells expressing CD8-GFP (n = 40 cells). (C) Maximum projection of the posterior crista in a larva with some hair cells expressing 139-231-GFP, which fills the cell (n = 43 cells). (D) Same as B except the transgene being expressed is 139-231-GFP. No rescue of FM 4C64 labeling was observed in hair cells expressing 139-231-GFP (n = 33 cells). Size pubs in C and A are 5m, in D and B are 10m.(TIF) pgen.1007635.s004.tif (3.7M) GUID:?71A1D86D-24FB-4014-84D0-3E7A8465547C S5 Fig: Nuclear mCherry fluorescence will not correlate with GFP-tagged Tmc fluorescence. XY plots from the integrated denseness of nuclear mCherry fluorescence vs the integrated denseness of GFP-tagged Tmc order Abiraterone fluorescence in the package area of lateral cristae. We analyzed 4 dpf larvae. (A) Package ideals for constructs Compact disc8-2TM and 97C113 will be the identical to those reported in Fig 8H using co-expression with Tmc2b-GFP. Package ideals for the full-length Tmie create are the identical to those reported in Fig 4C using co-expression with Tmc1-GFP. (B) Package values will be the identical to those reported in Fig 8 using co-expression of every individual build with Tmc2b-GFP. We performed linear.