Figure 3(A) Cell count-based comparison of cellular proliferation of T-ALL cells after treatment with dexamethasone (Dexa). Individual and mean values of three biological replicates ± SD analyzed by two-way ANOVA are shown. Data information: For all displayed experiments dimethyl sulfoxide (DMSO; was used as a solvent control. ns = not significant, * p < 0.05, ** p < 0.01, *** p < 0.001.(B) Flow cytometric analysis of cell death by AnnexinV staining in T-ALL cell lines upon treatment with indicated Dexa concentrations. Cell death is represented by the number of AnnexinV positive (AnnexinV+), apoptotic cells as a percentage (%) of total cells. Mean values of n≥4 biological replicates ± SD are shown. Data information: ns = not significant, * p < 0.05, ** p < 0.01, *** p < 0.001.(C) Expression levels of human GRα and LRH-1 mRNA in human T-ALL cell lines were determined by probe-based real-time quantitative PCR, calculated as relative expression compared to beta-actin and used to determine the GR:LRH-1 ratio (right). Individual and mean values of four biological replicates ± SD are shown and statistically significant differences between CEM-C7 and GC-resistant cell lines were determined by one-way ANOVA.(D) Immunoblots of GR, BimEL, BimL and BimS and Tubulin from T-ALL cells treated for 24 h with 20 µM 3d2 or 30 µM Dexa. Representative results from n=2 biological replicates are shown. Data information: For all displayed experiments dimethyl sulfoxide (DMSO; represented by Ø was used as a solvent control. ns = not significant, * p < 0.05, ** p < 0.01, *** p < 0.001.(E) Immunoblots of GR, PARP (nuclear loading control) and Tubulin (cytoplasmatic loading control) from cytoplasmic (CP) and nucleoplasmic (NP) lysates of T-ALL cells treated with Dexa. Note: Nuclear extracts were more highly concentrated (approx. 6-fold) than cytosolic fractions. Representative results from two biological replicates are shown.(F) GR activity in human T-ALL cells transfected with a control luciferase reporter plasmid (pGL3) (F) GR responsive (GRE) luciferase reporter and treated with 3d2 and/or Dexa. β-galactosidase (bGal) was co-transfected as an internal transfection control. Luciferase reporter activity was normalized to bGal activity and calculated as relative to the DMSO-treated pGL3 control. Individual and mean values analyzed by two-way ANOVA of three biological replicates ± SD are shown for Jurkat and CEM-C7 whereas technical triplicates ± SD of a representative experiment (n=3 biological replicates) are shown for MOLT-4. Data information: For all displayed experiments dimethyl sulfoxide (DMSO; was used as a solvent control. ns = not significant, * p < 0.05, ** p < 0.01, *** p < 0.001.(G) LRH-1 activity in human T-ALL cells transfected with a control luciferase reporter plasmid (pGL3) and (G) LRH-1 responsive (5xRE) luciferase reporter and treated with 3d2 β-galactosidase (bGal) was co-transfected as an internal transfection control. Luciferase reporter activity was normalized to bGal activity and calculated as relative to the DMSO-treated pGL3 control. Individual and mean values analyzed by two-way ANOVA of three biological replicates ± SD are shown for Jurkat and CEM-C7 whereas technical triplicates ± SD of a representative experiment (n=3 biological replicates) are shown for MOLT-4. Data information: For all displayed experiments dimethyl sulfoxide (DMSO; was used as a solvent control. ns = not significant, * p < 0.05, ** p < 0.01, *** p < 0.001.(H) Flow cytometry-based quantification of BiFC in Jurkat cells transiently co-transfected with YFP1-LRH-1 and GR-YFP2 and treated for indicated time periods with Dexa. YFP+ cells are shown as a percentage (%) of viable cells. Individual and mean values ± SD of three biological replicates analyzed by one-way ANOVA are shown. Data information: For all displayed experiments dimethyl sulfoxide (DMSO; represented by 0h) was used as a solvent control. ns = not significant, * p < 0.05, ** p < 0.01, *** p < 0.001.