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. Author manuscript; available in PMC: 2013 Jun 5.
Published in final edited form as: Cell. 2012 Nov 21;151(5):937–950. doi: 10.1016/j.cell.2012.10.035

Figure 4. MED12 Suppresses TGF-β Signaling by Negatively Regulating TGF-βR2.

Figure 4

(A–F) MED12KD leads to induction of a panel of TGF-β target genes and EMT marker genes. mRNA expression analysis by qRT-PCR of TGF-β target genes ANGPTL4 (A), TAGLN (B), CYR61 (C), and CTGF (D) and EMT marker genes VIM (E) and CDH2 (F) in H3122 and PC9 cells expressing pLKO controls or shRNAs targeting MED12 is shown. Cells were cultured in normal condition without TGF-β stimulation. Error bars denote SD.

(G and H) MED12KD results in strong induction of TGF-βR2 protein and SMAD2 phosphorylation. Western blot analysis of H3122 (G) and PC9 (H) cells expressing pLKO or shMED12 vectors. HSP90 was used as a loading control.

(I) MED12KD results in a modest induction of TGF-βR2 mRNA in a time course experiment. RNA samples from PC9 cells expressing pLKO or shMED12 were collected at days 3, 5, and 8 post lentiviral infection, and TGF-βR2 mRNA was analyzed by qRT-PCR. Error bars denote SD.

(J) There is a progressive increase in TGF-βR2 protein levels in time after MED12KD, and the increase is associated with increased p-MEK, p-ERK, and N-cadherin. Western blotting analysis of the total lysates from the PC9 cells described in (I). All cells were treated with 25 nM gefinitib for 6 hr before lysate collection.

(K) MED12 localizes to both nucleus and cytoplasm. Western blotting analysis of the nuclear and cytoplasmic fractions prepared from PC9 cells expressing control vector or shMED12 with or without 16 hr of 25 nM gefitinib treatment. Lamin A/C and SP1 were used as marker controls for nuclear fractions, whereas α-TUBULIN and HSP90 were used as controls for cytoplasmic fractions.

(L) MED12 is capable of physically interacting with TGF-βR2. Western blotting analysis of coimmunoprecipitation experiments using Phoenix cells cotransfected with TGF-βR2 and MED12 in a ratio of 5:1 is shown.

(M) Cytoplasmic MED12 interacts with TGF-βR2 in PC9 cells. Western blotting analysis of coimmunoprecipitation experiments with a cytoplasmic fraction of parental PC9 cells is shown.

See also Figures S5 and S6.