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. Author manuscript; available in PMC: 2016 Dec 1.
Published in final edited form as: Exp Hematol. 2015 Sep 11;43(12):1031–1046.e12. doi: 10.1016/j.exphem.2015.08.013

Figure 4. Loss of FANCA disrupts spindle microtubule assembly at prometaphase centrosomes.

Figure 4

(A) Experiment design. Microtubules of living FANCA−/− and FANCA+ cells were destabilized by cold treatment (4°C for 1 hour). Cells were then returned to 37°C to initiate microtubule reassembly and fixed with 4% paraformaldehyde 15 seconds later. (B) Cold treatment fully destabilizes microtubules in FANCA+ and FANCA−/− prometaphase cells. (C) Representative images of mitotic spindle assembly in FANCA+ and FANCA−/− prometaphase cells stained with anti-α-tubulin (green) and anti-pericentrin (red) antibodies. Images were captured with 60× lens on the Deltavision deconvolution microscope. Scale bars: 2µm (left and right) and 500nm (region of interest showed in the center). (D, E) Quantification of spindle microtubules per centrosome (D) and the microtubule length (µM) (E) in gene-corrected and FANCA−/− cells treated as described in (A). Data represents 2 independent experiments (n=130 microtubules/experiment), and error bars represent SEM. (E) Representative mitotic HeLa cell stained with anti-FANCA (red) and anti-α-tubulin (green) antibodies, imaged on an ELYRA PS.1 super-resolution microscope using SIM technology. Insert shows enlarged centrosome-containing region of interest. White line shows the line of fluorescence intensity profile. Scale bar: 2µm. Cells were imaged with deconvolution microscopy (Applied Precision personalDx) and deconvolved with Softworx imaging suite (10 iterations, ratio: conservative). (F) Fluorescence intensity profiles of FANCA (red) and α-tubulin (green) signal.