XB-IMG-171614
Xenbase Image ID: 171614
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Figure S1 (related to Figures 1,2,3 and 4): Nup188 and Nup93 are required for cilia in
mammalian cells and Xenopus.
(A) Nup62 and Nup133 protein levels after MO injection. Western blots of Nup62 and
Nup133 in both uninjected controls (UCs) and embryos injected with nup62 and nup133 MOs
(nup62 MO doses: 5 and 10ng/embryo) (nup133 MO doses: 2.5, 5 and 10ng/embryo). Numbers
above each lane indicate the band intensity normalized to Gapdh (loading control). Boxed lanes
indicate the dose used in epidermal cilia stain in Figure 3B.
(B) Nup188 is required for cilia in human RPE cells. Merged (green and blue channels)
fluorescent images of human RPE cells induced to ciliate after knockdown of Nup188 or Nup93
using specific siRNAs or scrambled (mock) control. Cilia are stained with an Arl13b antibody
(green) and DNA with Hoechst (blue). See also Figure 4F and 4J for degree of knockdown.
(C) Nup188 depletion in RPE cells leads to decreased ciliation. Plot of percentage of ciliated
cells after knockdown of Nup188 or Nup93 using specific siRNAs in human RPE cells. n=total
number of nuclei/cilia from 3 independent experiments. Chi-Square Test, *** p<0.0005.
(D-F) Loss of nup93 affects gliding in Xenopus embryos.
(D) At stages 28-31, wildtype embryos glide along the surface of an agarose-coated dish due
to the beating of multiciliated cells while nup93 morphants do not. Brightfield images of 2 UCs
(top) and 4 nup93 morphant embryos (bottom).
(E) Same field as (D) but nup93 morphants are identified by Alexa488 tracer dye (bottom 4
green morphants).
(F) Video frame captures of the same UC and morphant embryos in (D) and (E) gliding at
indicated times. See also Movie S1.
(G) Nup93 is specifically required for epidermal cilia. Plot of the percentage of embryos with
abnormal epidermal cilia assessed by anti-AcTub staining after injection at 1-cell stage with
nup93 MO (1 ng/embryo) or (to assess specificity) with nup93 MO combined with human (h)
NUP93 mRNA (50 or 100 pg/embryo). Bottom panel is a key (blue, green, orange, red) to our
qualitative assessment of cilia shown as example fluorescent images of embryos (lateral views,
dorsal at top) stained with anti-Actub (green) and Hoechst (blue). n=total number of embryos
from 3 independent experiments, Chi-Square Test, *** p<0.0005.
(H-I) Cilia loss in nup morphants is not due to changes in cilia cell type specification.
(H) In situ hybridizations images with a foxj1-specific probe of stage 10.5 embryos injected
with the indicated MOs or with Xtnup188 mRNA. Vegetal views of embryos with blastopore lip to
the top are shown as representative examples of 2 independent experiments. Numbers indicate
the number of embryos showing the same staining pattern.
(I) In situ hybridizations images with a foxj1-specific probe of stage 28-31 embryos injected
with the indicated MOs. Left, lateral views of embryos with dorsal at the top are shown as
representative examples of 3 independent experiments.
(J) Nup188 and Nup93 depletion affects multiciliated cells in Xenopus epidermis.
Fluorescent images of epidermal animal caps derived from UC embryos or those injected with
the indicated MOs; multiciliated cells (MCCs) labeled with anti-AcTub (green). Two z-sections
(top and bottom panels) at the indicated focal planes are shown Image published in: Del Viso F et al. (2016) Copyright © 2016. Image reproduced with permission of the Publisher, Elsevier B. V.
Image source: Published
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