Figure 4. SMP30 Promoter in response to T3.
Transient transfections of hSMP30 TRE1, TRE2 (A, B, E, F, G, H) and mut hSMP30 TRE1 and TRE2 (C, D) were carried out using MCF-7 cells and similar experiments were also carried out in HEK 293T cell line as shown in Fig.S3. 20 hrs before transfection, cells were plated in DMEM 10%CS media, at a density of 1×105 cells per well in 12 well plates. For transient transfection, 0.5 µg of reporter plasmid DNA, 0. 5 µg of TRβ and TRα (TRs), RXRα expression vector or pCMV vector in (A,B,C,D,E,F) , 0.5 µg of only TRβ or TRα expression vector or pCMV vector in (G, H) ,50 ng of pRL-TK control vector were co transfected using Fugene HD transfection reagent (from Roche) as per manufacture's instruction. After 2 4hrs of transfection, cells were subjected to overnight treatment with 1 µM concentrations of T3 (A, B, C, D, G, H), 10 nM of E2, 1 µM of T3 or ethanol vehicle to cells (E, F) in 10% CS –DMEM. Then cell lysates were prepared and luciferase activities were measured. Values are the mean of three independent experiments ± SD normalized to Renilla activity. * P<0.0232 difference from control using ANOVA for Fig A, *** P<0.0001difference from vehicle control using ANOVA in Fig. B, C, D, E, F and G, ** P<0.001difference from vehicle control using ANOVA in Fig. H.