Figure 5. Mitotic spindle assembly is severely impaired in erythroblasts when centrosomes or CDK5RAP2 are absent.

• A
  Immunofluorescence images of untreated (untx) or centrinone‐B (CB)‐treated cells at 36 h (T36) of ex vivo culture. Representative examples for different mitotic spindle morphologies are shown. Cells were stained for α‐tubulin (magenta), γ‐tubulin (grey), phospho‐histone H3 (pHH3, green), and DNA (Hoechst, blue). Images are maximum intensity projections of deconvolved z‐stacks. Scale bar, 2 μm.
• B
  Quantification of mitotic spindle morphologies in untreated (untx) or centrinone‐B (CB)‐treated cells at T24 or T36 of ex vivo culture. Graph depicts percentage of spindle phenotypes. In total, 315 (untx, T24), 367 (CB, T24), 295 (untx, T36), and 282 (CB, T36) cells were analyzed from three litters.
• C
  Immunofluorescence images of Cdk5rap2^HET and Cdk5rap2^null cells at 36 h (T36) of ex vivo culture. Representative examples for different mitotic spindle morphologies are shown. Cells were stained for α‐tubulin (magenta), γ‐tubulin (grey), pHH3 (green), and DNA (Hoechst, blue). Images are maximum intensity projections of deconvolved z‐stacks. Scale bar, 2 μm.
• D
  Quantification of mitotic spindle morphology at T24 or T36 of ex vivo culture. Graph depicts percentage of spindle phenotypes. At T24, three Cdk5rap2^WT (323 cells), three Cdk5rap2^HET (296 cells), and two Cdk5rap2^null (197 cells) embryos were analyzed. At T36, five Cdk5rap2^WT (387 cells), six Cdk5rap2^HET (384 cells), and three Cdk5rap2^null (202 cells) embryos were analyzed.
• E
  Schematic (left) shows experimental outline for nocodazole treatment of ex vivo cultured erythroid progenitors at indicated time points. Representative ImageStream images of ex vivo cultured cells at different mitotic stages (right). Cells were stained for phospho‐Histone H3 (pHH3, green), TER119 (erythroid marker, magenta), and DNA (Hoechst, blue). BF: bright field. Scale bar, 10 μm.
• F, G
  Quantification of pHH3^pos, TER119^pos, cells and mitotic stages (see text for details) after 36 h (T36) upon DMSO or nocodazole treatment according to (E) using ImageStream. Number of embryos analyzed is shown in brackets.

Data information: Box plots show 5^th and 95^th (whiskers) and 25^th, 50^th, and 75^th percentiles (boxes). Bar graphs display mean ± s.d. Statistical analysis was based on the number of litters (B) or number of embryos (D, F, and G). Statistical significance was determined by one‐way ANOVA with Tukey's multiple comparisons test (B, D, F, and G). *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.