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Fig. 1. The Tgfß pathway is regulated by RA at the onset of lung
development. (A) Western blotting of cultured mouse foregut
explants (E8.5 plus 24 hours) revealing increased phosphorylation of Smad2
(pSmad2) in RA-deficient conditions (lane 3, BMS493-treated WT; lane 4,
non-RA-supplemented Raldh2-/-) as compared with
RA-sufficient conditions (lane 2, WT control; lane 5, RA-supplemented
Raldh2-/-). TGFß1-treated WT foregut is used as a
positive control (lane 1). Total Smad2 (tSmad2) is used for normalization.
(B) Real-time PCR showing rapid dose-dependent induction of
Tgfbi in TGFß1-treated lung mesenchymal cells (MLg cells;
asterisks indicate P<0.05 by Student's t-test).
(C,G) Hematoxylin and Eosin (H&E) staining of
paraffin-embedded sections showing lung bud formation in the control, but not
in the BMS493-treated foregut culture. (D,H) RARElacZ
expression is strong in control foregut cultures, but is dramatically
suppressed by BMS493 treatment (asterisk marks the presumptive lung region in
G,H). (E,F,I,J) Whole-mount in situ hybridization
(WMISH) and immunostaining of control foreguts revealing a low level of
Tgfbi mRNA (E, arrowheads) and Tgfbi protein (F, red) expression in
the foregut mesoderm of the lung primordium. BMS493 treatment results in
increased Tgfbi mRNA (I, arrowhead) and protein (J, red) in the
mesoderm of the presumptive lung and stomach fields, and stronger pSmad2
signals, as compared with the control (F,J, green). Ht, heart; Lu, lung; St,
stomach; Ctr, control. Scale bar: 300 µm in E.
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