Journal: bioRxiv
Article Title: Loss of coiled-coil protein Cep55 impairs abscission processes and results in p53-dependent apoptosis in developing cortex
doi: 10.1101/2020.06.02.129346
Figure Lengend Snippet: (A) Representative images of cortical sections from Cep55 +/− and −/− E14.5 embryos immunostained for neuron marker Tubb3 (gray) show cortical plate (cp) and axons in the intermediate zone (iz). In −/− images, the cp is disorganized the border between cp and iz is unclear. (B) Mean thicknesses of total cortex and neuron layer are significantly decreased in −/− brains. (C) The −/− cp/iz occupies proportionally less of cortical width than normal, while the vz/svz occupies more . (D) Cortical sections stained for NSC marker Pax6 (red) and basal progenitor (BP) marker Tbr2 (green) show NSCs in the vz of Cep55−/− brains are more disorganized, with some empty spaces (square), and some nuclei mislocalized basally above the svz (arrows). (E-F) NSCs (Pax6+) per cortical length are reduced in Cep55−/− and some mislocalized. BP numbers (Tbr2+) are not significantly changed. For B, total thickness, n = 7 +/− and 8 −/− brains. For B, cp/iz and vz/svz thickness, and for C,E,F, n= 4 +/− and 4 −/− brains. (G) Phospho-histone H3 (PH3) immunostaining is used to mark cells in mitosis. (H) Cep55−/− cortices show a normal number of mitotic cells (PH3+, magenta) at the apical membrane but an increased number of mitotic cells basally. (I) The mitotic index of NSCs (Pax6+) is normal, but of BPs (Tbr2+) is significantly increased in Cep55 −/− cortices. Dashed line in A,D,G = apical membrane. For H,I: n= 4 Cep55+/− and 4 −/− brains. Scale bar: (A,G): 20 μm. n.s.; not significant, * p < 0.05; ** p < 0.01; *** p < 0.001. All experiments, Student’s t-test.
Article Snippet: Antibodies used in this analysis: mouse monoclonal anti-mouse citron kinase (1:100, CITK; 611367 BD Biosciences, San Jose, CA), mouse polyclonal anti-human Cep55 (1:00, H00055165-B01P Abnova, Taipei, Taiwan), mouse monoclonal anti-mouse Cep55 (1:100 for immunofluorescence experiments, sc-377018 Santa Cruz,, Dallas, Texas), rabbit polyclonal anti-human CC3 (1:250, 9661s Cell-Signaling, Danvers, MA), rat monoclonal anti-mouse Tbr2 (1:200, 14-4875, eBioscience (Thermo Fisher Scientific), Waltham, MA), rabbit polyclonal anti-mouse Pax6 (1:200, PRB-278P, BioLegend, San Diego, CA), mouse monoclonal anti-rat Aurora B kinase (1:300, 611082 BD Biosciences, San Jose, CA), rabbit monoclonal anti-human Aurora B kinase (1:100, ab2254, Abcam, Cambridge, MA), rat monoclonal alpha-tubulin (1:300, NB600-506, Novus Biologicals, Centennial, CO), rabbit monoclonal anti-human PH3 (1:200, 3458 Cell Signaling, Danvers, MA), chicken polyclonal anti-mouse Nestin (1:600, NES, Aves Labs, Davis, CA), rat monoclonal anti-human Ki67 (1:100, 14-5698 eBioscience, Waltham, MA), rabbit polyclonal anti-mouse p53 (1:500, NCL-L-p53-CM5p Leica Biosystems, Wetzlar, Germany), rat monoclonal anti-human Ctip2 (1:400, 18465 Abcam, Cambridge, MA), rabbit polyclonal anti-mouse Tbr1 (1:200, 31940 Abcam, Cambridge, MA), rabbit monoclonal Satb2 (1:200 Ab92446 Abcam, Cambridge, MA), rabbit polyclonal pericentrin (1:500, 92371, BioLegend, San Diego, CA), mouse monoclonal Tubb3 (Tuj1) (1:500, 801201, BioLegend, San Diego, CA), mouse monoclonal phospho-histone H3 (Ser10) (1:200, 9706, Cell Signaling, Danvers, MA), Phalloidin Oregon Green or 568 (1:50, 07466, Al2380 Invitrogen, Waltham, MA), chicken polyclonal anti-human alpha-tubulin (1:100, ab89984 Abcam, Cambridge, MA), mouse monoclonal Alix (1:100, SC-53538 Santa Cruz, Dallas, Texas), mouse monoclonal Tsg101 (1:100, SC-7964 Santa Cruz, Dallas, Texas), rabbit polyclonal CHMP2A (1:100, 10477-1-AP Proteintech, Chicago, IL), rat monoclonal Zo-1 (1:50, R26.4DC, DSHB, Iowa City, IA) and polyclonal rabbit anti Zo-1 (1:50 61-7300, rabbit, Invitrogen (Thermo Fisher Scientific), Waltham, MA).
Techniques: Marker, Staining, Immunostaining
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