Tgfβ1

HL-1 cells were plated in 96-well plates and were transiently transfected with 0.15 μg of pGL4.48[luc2P/SBE/Hygro] vector (Promega, Madison, WI, USA) and 0.015 μg of Renilla luciferase control reporter vector (Promega, Madison, WI, USA). The transfection assay above was carried out with FuGENE six transfection reagents (Promega, Madison, WI, USA) following the manufacturer’s instructions. The SBE in pGL4.48 vector was the Smad binding element and luciferase activity was normalized to Renilla activity. After 24 h of transfection, HL-1 cells were treated with TGFβ1 (20 ng/ml, Peprotech, Rocky Hill, CT, USA) for 24 h. In the RUNX1 treated group, cells were treated with TGFβ1 (20 ng/ml) for 12 h and then co-treated with recombinant RUNX1 protein (Origene Technologies, Rockville, MD, USA) for 12 h. In the end, cells were collected and put into luciferase reporter assay.

The long isoform of mouse LTBP4-L (uc009fvt.2, transcript variant 1) was ligated into the expression vector pcDNA3.1/V5-His-TOPO (Invitrogen) and engineered to include an Xpress epitope tag in frame at the immediate carboxy terminus. Mouse cDNA clones of Mstn (NM_010834), Gdf11 (NM_010272), Tgfβ1 (NM_011577), Tgfβ2 (NM_009367), and Tgfβ3 (NM_009368) were purchased from Origene (catalog numbers MR227629, MR223819, MR227339, MR225633, and MR206441, respectively). A myc-DDK tag was included in frame at the extreme carboxy teriminus (3’) end of each open reading frame. Full length human LTBP4 (NM_001042544.1), MSTN (NM_005259.1), and myc-tagged GDF11 (NM_005811) cDNA clones were purchased from Origene (catalog numbers SC311430, SC124056, and RC222080, respectively). The Xpress epitope (DLYDDDDK) was added onto the 5’/N-terminus of human LTBP4. Primers encoding the Myc epitope tag (EQKLISEEDL) were used to add this sequence to the 3’/C-term of MSTN. Amino and carboxy terminal clones corresponding to exons 1–12 and exons 11–34 of human LTBP4 were cloned and engineered to contain Xpress tags on the amino termini. Schematics of the constructs are shown in S11 Fig.

To study Slug overexpression, 1 x 10⁶ cells were transfected with XL6 plasmid and XL6-Slug using the Amaxa Biosystem (Lonza) following manufacturer’s guidelines. One day after electroporation, cells were cultured with starved medium ON and stimulated with 10 ng/ml of TGFβ1 (Acris) for 24 h.
Slug knockdown was accomplished with 10nM of siPool against Slug (SiTool Biotech GmBH, Martinsried), which allows the use of low concentrations of siRNA minimizing off-targets effects [23 (link)]. A scrambled sequence was used as control. Both siPools were transfected using Lipofectamine RNAimax (Invitrogen).