Journal: Journal of the Endocrine Society

Article Title: Functional Characterization of Glucocorticoid Receptor Variants Is Required to Avoid Misinterpretation of NGS Data

doi: 10.1210/js.2019-00028

Figure Lengend Snippet: Dose-response curves of transcriptional activation of the GR variants compared with that of the WT receptor. (A) HEK293T cells plated in 96-well plates were cotransfected with 40 ng of plasmid encoding GR WT or GR variants (F65V, M86V, A229T, A304E, N374S, and R386Q), together with the reporter luciferase plasmid pMMTV-Luc, in which luciferase activity is under the control of GR response elements, and pMIR- β -gal encoding β -galactosidase. After 24-h stimulation by ethanol or increasing concentrations of DXM (10 −10 to 10 −6 M), enzymatic activities were measured. Transcriptional activities are expressed relative to the maximum transactivation of the WT (arbitrary set at 1). Each point represents the mean ± SEM of at least 30 replicates performed from at least three independent experiments. Representation was fitted with the “log(agonist) vs response three parameters” function of GraphPad software. (B) Comparison of EC50 (DXM concentration inducing half-maximum transactivation) and of maximum transcriptional activity (B max ) of the six GR variants compared with the WT. Values are mean ± SEM, obtained from four independent experiments (nonparametric ANOVA Kruskal-Wallis tests followed by Dunn posttests). (C) The absence of dominant negative effects of the variants on the WT is shown. HEK293T cells plated in 96-well plates were cotransfected with 40 ng of plasmid encoding GR WT or 20 ng of plasmid encoding WT plus 20 ng encoding GR variants (F65V, A304E, and N374S), together with the reporter luciferase plasmid pMMTV-Luc, in which luciferase expression is under the control of GR response elements, and pMIR- β -gal coding β -galactosidase. After 24-h stimulation by ethanol or increasing concentrations of DXM (10 −10 to 10 −6 M), enzymatic activities were measured. Transcriptional activities are expressed relative to the maximum transactivation of the WT (arbitrary set at 1). Each point represents the mean ± SEM of at least 30 replicates performed from at least three independent experiments. Representation was fitted with the “log(agonist) vs response three parameters” function of GraphPad software. EC50, ligand concentration leading to 50% maximal transactivation capacity.

Article Snippet: Embryonic human kidney HEK293T and African green monkey kidney COS-7 cells were grown in DMEM (Life Technologies, Saint-Aubin, France) with 10% fetal calf serum (Biowest, Nuaille´, France) and penicillin/streptomycin (GE Healthcare, Vélizy-Villacoublay, France) and were incubated at 37°C in a humidified atmosphere containing 5% CO 2 [ ].

Techniques: Activation Assay, Plasmid Preparation, Luciferase, Activity Assay, Software, Concentration Assay, Dominant Negative Mutation, Expressing

Journal: Journal of the Endocrine Society

Article Title: Functional Characterization of Glucocorticoid Receptor Variants Is Required to Avoid Misinterpretation of NGS Data

doi: 10.1210/js.2019-00028

Figure Lengend Snippet: Protein expression of the WT and the six GR variants. (A) HEK293T cells were transiently transfected or not with a plasmid encoding the WT or the six GR variants. Twenty-four hours posttransfection, protein extracts (20 µg) were analyzed by western blot followed by immunostaining with an antibody recognizing the NTD of GR (29; dilution 1/500) and an anti‒ β -actin antibody (30; dilution 1/1000). GR α protein expression was similar between the WT and the six variants but was below the detectable threshold in the untransfected cells used as control ( i.e., C). Representative image of three independent experiments is shown. The molecular weight marker used was PageRuler TM (Thermo Fisher Scientific). (B) Quantification of the protein signal did not reveal any statistically significant difference between the WT and the GR variants. Results are expressed as means ± SEM.

Article Snippet: Embryonic human kidney HEK293T and African green monkey kidney COS-7 cells were grown in DMEM (Life Technologies, Saint-Aubin, France) with 10% fetal calf serum (Biowest, Nuaille´, France) and penicillin/streptomycin (GE Healthcare, Vélizy-Villacoublay, France) and were incubated at 37°C in a humidified atmosphere containing 5% CO 2 [ ].

Techniques: Expressing, Transfection, Plasmid Preparation, Western Blot, Immunostaining, Molecular Weight, Marker

Journal: Journal of the Endocrine Society

Article Title: Functional Characterization of Glucocorticoid Receptor Variants Is Required to Avoid Misinterpretation of NGS Data

doi: 10.1210/js.2019-00028

Figure Lengend Snippet: 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 . Cells were treated (+) or not (−) with 5 nM of DXM for 24 hours. Transcriptional activities were measured as in 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 . Cells were treated (+) or not (−) with 5 nM of DXM for 24 hours. Transcriptional activities were measured as in 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.

Article Snippet: Embryonic human kidney HEK293T and African green monkey kidney COS-7 cells were grown in DMEM (Life Technologies, Saint-Aubin, France) with 10% fetal calf serum (Biowest, Nuaille´, France) and penicillin/streptomycin (GE Healthcare, Vélizy-Villacoublay, France) and were incubated at 37°C in a humidified atmosphere containing 5% CO 2 [ ].

Techniques: Plasmid Preparation, Construct, Cotransfection, MANN-WHITNEY

Journal: eLife

Article Title: Interplay between PML NBs and HIRA for H3.3 dynamics following type I interferon stimulus

doi: 10.7554/eLife.80156

Figure Lengend Snippet:

Article Snippet: Human BJ primary foreskin fibroblasts (ATCC, CRL-2522), human IMR90 fetal lung fibroblasts (ATCC, CCL-186), human HEK 293T embryonic kidney cells (Intercell, AG) and mouse MEFs embryonic fibroblasts Pml +/+ or Pml -/- (from Dr. Lallemand-Breitenbach, and whose cell identity was authenticated by STR profiling) were cultivated in DMEM medium (Sigma-Aldrich, D6429) containing 10% of fetal calf serum (FCS) (Sigma-Aldrich, F7524), 1% of penicillin/streptomycin (Sigma-Aldrich, P4458), at 37 °C under 5% CO2 and humid atmosphere.

Techniques: Transduction, Recombinant, Plasmid Preparation, Clone Assay, Mutagenesis, Sequencing, Quantitative RT-PCR, Concentration Assay, In Situ, Software, ChIP-sequencing