Panel A: Fluorescent images showing the staining of Spc105 in green, CID in magenta, and DAPI in blue in prometaphase I cells from different genetic lines. Panel B: Scatter plot showing the relative fluorescent intensity of Spc105 per nucleus in prometaphase I cells. The y-axis represents the relative Spc105 intensity per nucleus, and the x-axis lists the genetic lines. Panel C: Fluorescent images showing the staining of CENP-C in green, HA in magenta, and DAPI in prometaphase I cells from CENP-C rescue flies. Panel D: Fluorescent images showing the staining of CENP-C in green, CID in magenta, and DAPI in prometaphase I cells from bam-GAL4 and CENP-C rescue testes. Panel E: Scatter plot showing the relative fluorescent intensity of CID per nucleus in prometaphase I cells from bam-GAL4 control and CENP-C rescue. The y-axis represents the relative CID intensity per nucleus, and the x-axis lists the genetic lines. Panel F: Scatter plot showing the relative fluorescent intensity of CENP-C per nucleus in prometaphase I cells from bam-GAL4 control and CENP-C rescue. The y-axis represents the relative CENP-C intensity per nucleus, and the x-axis lists the genetic lines. Panel G: Fluorescent images showing the staining of Spc105 in green, CID in magenta, and DAPI in prometaphase I cells from bam-GAL4 and CENP-C rescue testes. Panel H: Scatter plot showing the relative fluorescent intensity of Spc105 per nucleus in prometaphase I cells from bam-GAL4 and CENP-C rescue. The y-axis represents the relative Spc105 intensity per nucleus, and the x-axis lists the genetic lines. Panel I: Fluorescent images showing normal and abnormal microtubule spindles with tubulin-GFP in green, CID in magenta, and DAPI. Panel J: Bar graph showing the percentage of microtubule spindle abnormalities in each knockdown line with meiosis I and II pooled together. The y-axis represents the percentage of spindle abnormality, and the x-axis lists the genetic lines.
Outer kinetochore recruitment and microtubule spindle assembly in CID, CAL1, and CENP-C RNAi. (A) Pupal testes stained with anti-Spc105 (green) and anti-CID (magenta) antibodies, and DAPI (blue). Representative images of prometaphase I cells from each of the genetic lines (bam-GAL4, CID RNAi, CAL1 RNAi, and CENP-C RNAi). Scale bars = 5 µm. (B) Quantitation showing the relative fluorescent intensity per nucleus of the outer kinetochore component Spc105 (homolog of KNL1 in humans) at prometaphase I. n = 52, 32, 40, 57. 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. Median intensity is shown for CENP-C RNAi (23.5%). Error bars = SEM. (C) Prometaphase I cell from CENP-C rescue flies stained with anti-CENP-C (green), anti-HA (magenta), and DAPI. Scale bar = 5 µm. (D) Representative images of prometaphase I cells from bam-GAL4 and CENP-C rescue testes showing anti-CENP-C (green), anti-CID (magenta), and DAPI (blue). Scale bar = 5 µm. (E) Quantitation showing relative fluorescent intensity per nucleus of CID in bam-GAL4 control and CENP-C rescue cells at prometaphase I. n = 34, 36. (F) Quantitation showing relative fluorescent intensity per nucleus of CENP-C. n = 34, 36. (G) Representative images of prometaphase I cells from bam-GAL4 and CENP-C rescue testes showing anti-Spc105 (green), anti-CID (magenta), and DAPI (blue). Scale bar = 5 µm. (H) Quantitation showing relative fluorescent intensity per nucleus of Spc105. n = 35, 33. (I) Representative images of normal and abnormal microtubule spindles showing tubulin-GFP (green), anti-CID (magenta), and DAPI (blue). Scale bar = 5 µm. (J) Quantitation of microtubule spindle abnormality in each knockdown line with meiosis I and II pooled together. KNL1, Kinetochore Scaffold 1.