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. 2020 Sep 18;2(3):zcaa021. doi: 10.1093/narcan/zcaa021

Figure 2.

Figure 2.

Activation of TGF-β signaling under hypoxia causes exon 11a exclusion from hMENA pre-mRNA. Immunofluorescence of pSMAD2/3 showing enhanced nuclear localization under hypoxia as compared to normoxia in MCF7 (A) and HCC1806 (B) after 12 h. (C) qRT-PCR for hMENA11a isoform 24 h after TGF-β induction in MCF7 (Ct values normalized to RPS16). (D) Relative luciferase activity (SBE4:Luc) after 12 h of TGF-β (10 ng/ml) induction in MCF7 versus control. (E) qRT-PCR for hMENA11a after 24 h of TGF-β signaling inhibition under hypoxia in MCF7. (F) Relative luciferase activity (SBE4:Luc) after 12 h of TGF-β signaling inhibition in MCF7 using 30 μM LY-364947 inhibitor under hypoxia. (G) Dynamic light scattering analysis (done using conditioned media of hypoxia-treated HCC1806) showing particles with a size distribution of 20–200 nm. Most of the particles fall in the range defined for exosomes (40–100 nm). (H) Immunoblot for TGF-β in normoxic and hypoxic exosomes (CD63 exosomal marker was used as a control) collected from media of HCC1806 treated under normoxia and hypoxia, respectively. (I) Relative luciferase activity (SBE4:Luc) after 12 h of normoxia- and hypoxia-conditioned (CM) media treatment in HCC1806. (J) qRT-PCR for hMENA11a, 24 h after normoxia- and hypoxia-conditioned treatment in HCC1806. Error bars show mean values ± SD (n = 3 unless otherwise specified). As calculated using two-tailed Student’s t-test, *P < 0.05, **P < 0.01 and ***P < 0.001.