Fig 3. CDK2/Speedy A localization in Cdk2^T160A spermatocytes.

Chromosome spread preparations from adult (postnatal day 40) testes immunostained with CDK2 (green) and Speedy A (red) in conjunction with SYCP3 (blue) are shown for WT (A–D) and Cdk2^T160A (G–J) spermatocytes for selected stages of meiotic prophase I. CDK2 and Speedy A can be detected as colocalized foci at all telomeric ends for all meiotic stages in WT (A–D and insets E–F) and Cdk2^T160A spermatocytes (G–J and insets K–L). Interstitial CDK2 foci—absent of Speedy A positivity—marking LRNs are transiently observed in mid-pachytene–stage WT (D) and insets (E–F), but not Cdk2^T160A spermatocytes (J and insets K, L). X–Y bivalents (blue XY labels) were also found to be positive for both CDK2 and Speedy A from early-pachytene onwards in both genotypes. All images are representative of at least 20 images taken for specified stages. Similar staining patterns were confirmed in at least 3 biological replicates. In all main panels, scale bars are representative of 5 μm; in all inset pictures, scale bars are representative of 1.25 μm. Interstitial CDK2 foci (M) were quantified specifically for mid-pachytene stages by counting the average numbers of nontelomeric CDK2 foci counted per nucleus. Data are presented as individual foci counts for WT (orange bars, N = 72) and Cdk2^T160A (blue bars, N = 50); error bars are indicative of the mean and SD. For mid-pachytene, Cdk2^T160A spermatocytes (J) with decreased CDK2/Speedy A signal in a subset of nuclei was observed (as marked by red arrows in insets K, L). The average telomeric CDK2 focus intensity/nuclei were quantified specifically for mid-pachytene stages (N). Data are presented as a mean intensity in AU ± SD determined from 3 biological replicates (N = 48 overall nuclei counted for WT (orange bars) and N = 48 overall nuclei counted (blue bars) for Cdk2^T160A. Telomeric signals were only counted from autosomes and were excluded if involved in telomeric fusion events. All intensity values calculated for a single nucleus were normalized to the background intensity of that nuclei. Percentages of mid-pachytene–stage nuclei with at least one telomere showing a decrease in telomeric CDK2 intensity of ≥50% (as compared with the average telomeric CDK2 intensity for that cell) are quantified in panel O. Individual data used to make panel O are shown in S3G Fig. All data were assumed to be non-normally distributed. Statistical significance between genotypes was determined by unpaired t test. Significance and P-values are reported directly over each comparison. The underlying data for (M, N, O) can be found in S1 Data. AU, arbitrary unit; CDK2, cyclin-dependent kinase 2; LRN, late recombination nodule; SD, standard deviation; SYCP, synaptonemal complex protein; WT, wild-type.