Figure 5.

Effect of the coactivator SRC2 on GR transactivation. (A) HEK293T cells were plated on 96-well plates and cotransfected with 40 ng of plasmid encoding GR WT or the variants (F65V, M86V, A229T, A304E, and N374S), together with a plasmid encoding the coactivator SRC2 (40 ng/well) (+) as in the legend of Fig. 2. Cells were treated (+) or not (−) with 5 nM of DXM for 24 hours. Transcriptional activities were measured as in Fig. 2 and were expressed as mean ± SEM of 12 replicates from three independent experiments, normalized for each construct to the transactivation value obtained after stimulation by DXM in the absence of SRC2, arbitrarily set at 1. There was no difference between the induction of transactivation by SRC2 for the WT and all the variants, except for N374S, which showed no increase in transactivation following SRC2 cotransfection. ***P < 0.001, nonparametric ANOVA Kruskal-Wallis test followed by Dunn posttests. (B) HEK293T cells were plated on 96-well plates and cotransfected with 40 ng of plasmid encoding GR WT or the variants (M86V, N374E, and R386Q), together with a plasmid encoding the coactivator SRC2 (40 ng/well) (+) as in the legend of Fig. 2A. Cells were treated (+) or not (−) with 5 nM of DXM for 24 hours. Transcriptional activities were measured as in Fig. 2A and were expressed as mean ± SEM of eight to 12 replicates from three independent experiments, normalized for each construct to the transactivation value obtained after stimulation by DXM in the absence of SRC2, arbitrarily set at 1. SRC2 cotransfection stimulated transactivation of the WT as well as that of the M86V and R386Q variants but not that of N374S and N374E variants. ***P < 0.001, nonparametric Mann-Whitney U tests. ns, not significant.