Fig. 1. GRdim and GRmon display attenuated transcriptional regulation compared to GRwt.

a The parental and GRKO cell lines were derived from a C127 murine cell line (3617). The parental cell line contains endogenous GR while the GRKO has endogenous GR knocked out via CRISPR-Cas9. The Cartoon represents the GR mutants used in this study. The wild-type (GRwt) dimerizes through at least two surfaces, located in the ligand binding domain (LBD) and the Distal (D) loop within the DNA-binding domain (DBD). GRdim has a point mutation (A465T) which affects DBD-DBD contacts but remains dimeric through LBD-LBD interactions. GRmon has the A465T mutation as well as an LBD mutation and is therefore monomeric. The GRwt, GRdim, or GRmon of mouse GFP-GR were re-introduced separately into the GRKO cell line and constitutively expressed. b Western blots for GR. GAPDH is a loading control. M, molecular weight marker. Endogenous GR, GFP-GR and GAPDH bands are highlighted on the right with arrows; * denotes non-specific bands. GAPDH blot was repeated on a separate gel due to an air bubble during protein transfer (Supplementary Fig. 9). The absence of a GR band in the KO cell line was independently confirmed three times, while the expression levels of GR mutants were done twice. c Volcano-plot representation from RNA-seq data after 2 h Dex treatment. NS, non-significant. FC, fold change. FDR, false discovery rate. The dashed lines indicate the thresholds used (|FC|> 0.6, FDR < 0.001) and the different colors in each datapoint indicate whether they meet FC, FDR, or both criteria. d Venn diagram of differentially expressed genes from RNA-seq data after 2 h Dex treatment.