Panel A: Fluorescence microscopy images showing CENP-C, CID, and DAPI signals in spermatid T5 nuclei. Panel B: Scatter plot showing relative CID intensity per nucleus across RNA interference genotypes. Panel C: Fluorescence microscopy images showing CENP-C, CID, and DAPI signals during prometaphase I. Panel D: Scatter plot showing relative CID intensity per nucleus during prometaphase I across genotypes. Panel E: Fluorescence microscopy images showing CENP-C, CID, and DAPI signals in spermatid T5 nuclei. Panel F: Scatter plot showing relative CID intensity per nucleus in spermatid T5 stage cells. Panel G: Scatter plot showing relative CENP-C intensity per nucleus during prometaphase I. Panel H: Fluorescence microscopy images showing Spc105, CID, and DAPI signals during prometaphase I. Panel I: Scatter plot showing relative Spc105 intensity per nucleus during prometaphase I.
Requirements for the second phase of CID assembly after meiosis II. (A) Pupal testes stained with anti-CENP-C (green) and anti-CID (magenta) antibodies, and DAPI (blue). Representative images of T5 spermatid nuclei from each of the genetic lines (bam-GAL4, CID RNAi, CAL1 RNAi, and CENP-C RNAi). CENP-C staining is notably absent from each cell, verifying T5 stage spermatid identity. Scale bars = 3 µm. (B) Quantitation showing the relative fluorescent intensity per nucleus of CID. n = 32, 29, 27, 26. Here, and throughout this figure, n represents the number of cells analyzed from at least three independent replicates; data were checked for normality using Shapiro–Wilk tests and analyzed using either Welch’s or Mann–Whitney tests. Error bars = SEM. (C) Pupal testes from a new GAL4 driver, Rbp4-GAL4 (expressed between S1 and S6), crossed to each RNAi line. Representative images of prometaphase I cells stained with anti-CENP-C (green) and anti-CID (magenta) antibodies, and DAPI (blue) from each of the genetic lines (Rbp4-GAL4, CID RNAi, CAL1 RNAi, and CENP-C RNAi). Scale bars = 5 µm. (D) Quantitation showing the relative fluorescent intensity of CID per nucleus at prometaphase I. n = 50, 76, 57, 67. Error bars = SEM. (E) Representative images of early spermatid nuclei (T5 stage) stained with anti-CENP-C (green), anti-CID (magenta), and DAPI (blue) for each knockdown line and control (Rbp4-GAL4, CID RNAi, CAL1 RNAi, and CENP-C RNAi). CENP-C staining is absent, indicative of T5 stage spermatids. Scale bars = 3 µm. (F) Quantitation showing relative fluorescent intensity of CID per T5 nucleus. n = 39, 33, 32, 31. Error bars = SEM. (G) Quantitation showing the relative fluorescent intensity of CENP-C per nucleus at prometaphase I for each knockdown line and control (Rbp4-GAL4, CID RNAi, CAL1 RNAi, and CENP-C RNAi). n = 44, 39, 38, 39. Error bars = SEM. (H) Representative images of prometaphase I cells stained with anti-Spc105 (green) and anti-CID (magenta) antibodies and DAPI (blue) from the genetic lines Rbp4-GAL4 (control) and CENP-C RNAi. (I) Quantitation showing the relative fluorescent intensity of Spc105 per nucleus at prometaphase I. n = 45, 44. Error bars = SEM.