Panel A shows a Western blot with two lanes for each genotype: w1118, cep135 minus slash minus, EGFP-Ana1; cep135 minus slash minus, and a loading control with alpha-tubulin. The blot detects Cep135 protein levels. Panel B displays microscopy images of centrioles in primary spermatocytes stained for EGFP-Ana1 (green), Asl (red), and DNA (blue). Scale bars are 10 micrometers for the overview image and 2 micrometers for the insets. Panel C presents a dot plot showing the mean and standard deviation of centriole lengths in mature primary spermatocytes for each genotype. The x-axis represents different genotypes, and the y-axis represents centriole length in micrometers. Each point represents the average length measured from a 16-cell cyst. Panel D shows a bar graph depicting the mean and standard deviation of Ana1 mean fluorescence intensity per unit length of centriole for each genotype. The x-axis represents different genotypes, and the y-axis represents intensity per micrometer in arbitrary units. Each point represents the average intensity measured from a 16-cell cyst.
Cep135 is required for Ana1-induced centriole overelongation. (A) Western blot of extracts from 50 to 60 testes of indicated genotypes. Detection of Cep135 was performed using specific antibodies, with α-tubulin as a loading control for protein levels in lysates. (B) Centrioles in primary spermatocytes stained to reveal EGFP-Ana1 (green), Asl (red), and DNA (blue). Scale bars, 10 µm (overview image) or 2 µm (insets). (C) Graphs show the mean ± SD quantification for centriole lengths in mature primary spermatocytes, using Asl as a centriole marker for each genotype. Each point on the graph represents the average length measured from a 16-cell cyst with 10–15 centrioles scored; five independent testes were measured for each genotype with 2–3 cell cysts scored in each testis. ****P < 0.0001, **P < 0.01. (D) Graphs show the mean ± SD quantification of Ana1 mean fluorescence intensity (per unit length of centriole) for each genotype. Each point on the graph represents the average intensity measured from a 16-cell cyst with 10–15 centrioles scored; five independent testes were measured for each genotype with 2–3 cell cysts scored in each testis. ns, not significant using a two-tailed unpaired t test. Source data are available for this figure: SourceData F2.