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. 2015 Oct 14;106(11):1524–1533. doi: 10.1111/cas.12773

Figure 2.

Figure 2

Effect of hypoxia on transforming growth factor‐β (TGF‐β)/Smad signaling. (a) Effect of long‐term hypoxia on TGF‐β‐induced Smad2 phosphorylation. Lewis lung carcinoma (LLC) cells maintained under hypoxia for 14 days were stimulated with 5 ng/mL TGF‐β for 1, 2, 4, 8, 12, 16 and 24 h. These cells were lysed and subjected to western blot analysis using PS2 (upper panel) and anti‐Smad2 antibody (lower panel). (b) Quantification for TGF‐β‐induced Smad2 phosphorylation under long‐term hypoxia. The intensity of Smad2 phosphorylation (a; upper panel) was normalized using the intensity of the band corresponding to Smad2 (a; lower panel). Each relative intensity was calculated by comparing it with the value for cells which are treated with TGF‐β for 1 h under normoxia. (c) Comparison of TGF‐β‐mediated (CAGA)12‐luc reporter activity between normoxia and long‐term hypoxia. LLC cells maintained under hypoxia for more than 14 days were transiently transfected with the (CAGA)12‐luc reporter and stimulated with 5 ng/mL TGF‐β for 16 h. The value from cells in the presence of TGF‐β in each condition was normalized using that in the absence of TGF‐β in each condition. (d) Comparison of TGF‐β‐mediated (SBE)4‐luc reporter activity between normoxia and long‐term hypoxia. All experiments except for (SBE)4‐luc reporter instead of (CAGA)12‐luc reporter were carried out according to (c). (e) Effect of short‐term hypoxia on TGF‐β‐induced Smad2 phosphorylation. LLC cells maintained under hypoxia for 16 h were stimulated with 5 ng/mL TGF‐β for 1, 2, 4 and 8 h. These cells were lysed and subjected to western blot analysis using PS2 (upper panel) and anti‐Smad2 antibody (lower panel). (f) Quantification for TGF‐ β‐induced Smad2 phosphorylation under short‐term hypoxia. Normalization was performed according to (b). (g) Comparison of TGF‐β‐mediated (CAGA)12‐luc reporter activity between normoxia and short‐term hypoxia. LLC cells maintained under hypoxia for 16 h were transiently transfected with (CAGA)12‐luc and stimulated with 5 ng/mL TGF‐β for 16 h. Experiments were performed according to (c). (h) Comparison of TGF‐β‐mediated p5HRE‐luc reporter activity between normoxia and long‐term hypoxia. All experiments except for p5HRE‐luc instead of (CAGA)12‐luc were carried out according to (c).