Journal: Scientific Reports

Article Title: Novel role for receptor dimerization in post-translational processing and turnover of the GRα

doi: 10.1038/s41598-018-32440-z

Figure Lengend Snippet: Rate of hGRwt protein turnover is altered in a ligand-selective manner. COS-1 cells were seeded into a 24 well plate (5 × 10 4 cells/well) and transiently transfected the next day with hGRwt. Following 24 hours incubation, cells were treated with solvent (EtOH) or the GCs, Dex and F, or CpdA (10 −5 M) for 2 to 72 hours. Thereafter, whole cell GRα-binding ( A ) was conducted using 20 nM [ 3 H]-Dex. Once lysed, hGRwt levels were detected via a scintillation counter and specific binding values (cpm) were plotted against time. Specific binding values of lysates from solvent (EtOH) and treated cells were plotted for each time point. Whole cell GRα-binding results shown are representative of four independent experiments (average ± SEM), conducted in triplicate. Statistical analysis of logarithmically transformed data was conducted using a two-way ANOVA followed by a Bonferroni multiple comparisons post-test comparing each time point to solvent (EtOH) (ns, P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001). Half-lives ( B ) and rate constants ( C , actual values above bars) were calculated using non-linear regression one-phase dissociation decay analysis. For statistical analysis of rate constants, one-way ANOVA followed by a Dunnett’s multiple comparisons post-test was conducted on logarithmically transformed data comparing K (cpm/hour) values to solvent (EtOH) (ns, P > 0.05, ***P < 0.001).

Article Snippet: African green monkey kidney ( COS-1) cells and the human liver carcinoma cell line (HepG2), containing endogenous GRα, were purchased from American Type Culture Collection (USA).

Techniques: Transfection, Incubation, Solvent, Binding Assay, Transformation Assay

Journal: Scientific Reports

Article Title: Novel role for receptor dimerization in post-translational processing and turnover of the GRα

doi: 10.1038/s41598-018-32440-z

Figure Lengend Snippet: GRα dimerization is required for ligand-induced GRα protein turnover. COS-1 cells were seeded into a 24 well plate (5 × 10 4 cells/well) and transiently transfected the next day with hGRwt ( A and B ) or hGRdim ( C and D ). Following 24 hours incubation, cells were treated with Dex, F or CpdA (10 −5 M) for 2 to 72 hours. Thereafter hGRwt ( A ) and hGRdim ( C ) levels were monitored via whole cell GRα-binding, using 20 nM [ 3 H]-Dex. Specific binding of lysates from solvent (EtOH) treated cells was set at 100% (dotted line) for each time point and % specific binding of lysates from compound treated cells were then determined relative to solvent (EtOH), at each time point, and plotted. Whole cell GRα-binding results shown are representative of five independent experiments (average ± SEM), conducted in triplicate. Statistical analysis of logarithmically transformed data for ( A and C ) was conducted using a two-way ANOVA followed by a Bonferroni multiple comparisons post-test comparing experimental values to solvent (EtOH) (ns, P > 0.05, **P < 0.01, ***P < 0.001) or to Dex ( # P < 0.05, ## P < 0.01, ### P < 0.001). GRα protein levels of hGRwt ( B ) and hGRdim ( D ) were confirmed by Western blotting. The 24 hour treatment time point was repeated, with all ligands in the HepG2 cells (containing endogenous hGRα), which were seeded into a 12 well plate (5 × 10 4 cell/well). GRα levels were assessed using Western blotting where GAPDH was probed to ensure equal protein loading. Western blots shown are representative of three independent experiments ( E , inset). For quantification ( E ), the intensity of the hGRα and GAPDH bands was determined using UNSCANIT and hGRα levels were then normalized to GAPDH levels and expressed as a percentage (average ± SEM) of hGRα levels in the presence of the solvent (EtOH), which was set at 100% (dotted line). Statistical analysis of logarithmically transformed data for ( E ) was conducted using a one-way ANOVA with Dunnett’s multiple comparisons post-test comparing experimental values to solvent (EtOH) (ns, P > 0.05, *P < 0.05, ***P < 0.001). Full-length blots are presented in Supplementary Figure .

Article Snippet: African green monkey kidney ( COS-1) cells and the human liver carcinoma cell line (HepG2), containing endogenous GRα, were purchased from American Type Culture Collection (USA).

Techniques: Transfection, Incubation, Binding Assay, Solvent, Transformation Assay, Western Blot

Journal: Scientific Reports

Article Title: Novel role for receptor dimerization in post-translational processing and turnover of the GRα

doi: 10.1038/s41598-018-32440-z

Figure Lengend Snippet: Ligand-induced GRα protein turnover occurs predominantly via the proteasome. COS-1 cells were seeded in a 12 well plate (5 × 10 4 cell/well) and transiently transfected the next day with either hGRwt ( A and B ) or hGRdim ( A and C ). HepG2 cells that contain endogenous hGR were used in ( D ). Following 24 hours incubation, cells were treated with solvent (DMSO) or 1 µM proteasome inhibitor (MG132) for 1 hour and then, in the absence (−MG132) or presence of MG132 (+MG132), treated with solvent (EtOH) or the compounds Dex, F and CpdA (10 −5 M) for 16 hours. GRα protein levels were assessed by Western blotting, where GAPDH was probed to ensure equal protein loading. The Western blots shown ( B – D inset) are representative of two to three independent experiments. For quantification ( A – D ), the intensity of the GRα and GAPDH bands was determined using UNSCANIT and then the GRα levels were normalized to GAPDH levels and expressed as a percentage (average ± SEM) and plotted. Firstly, the effect of MG132 (+MG132) on unliganded GRα protein levels was investigated ( A ) and compared to GRα levels in the absence of MG132 (−MG132). To compare the effect MG132 treatment on unliganded GRα protein levels one-way ANOVA with a Tukey’s multiple comparisons post-test was conducted on logarithmically transformed data (ns, P > 0.05, **P < 0.01). Thereafter, the effect of MG132 (+MG132) on the extent of hGRwt ( B ), hGRdim ( C ) and endogenous hGRα ( D ) turnover was investigated. The dotted line, on all graphs, represents the fold increase in GRα levels in the presence of solvent (EtOH) and/or absence of MG132 (−MG132) and is set at 1-fold or 100%. To evaluate the effects of ligands on GRα levels in the absence (−MG132) and presence (+MG132) of MG132, statistical analysis was conducted on logarithmically transformed data using a one-way ANOVA with a Dunnett’s multiple comparisons post-test comparing to control (−MG132, EtOH) (ns, P > 0.05; # P < 0.05; ## P < 0.001). To analyse the significance of adding MG132 on the extent of ligand-induced GRα protein turnover, a two-way ANOVA was used on logarithmically transformed data followed by a Bonferroni post-test (ns, P > 0.05, **P < 0.01, ***P < 0.001).

Article Snippet: African green monkey kidney ( COS-1) cells and the human liver carcinoma cell line (HepG2), containing endogenous GRα, were purchased from American Type Culture Collection (USA).

Techniques: Transfection, Incubation, Solvent, Western Blot, Transformation Assay, Control

Journal: Scientific Reports

Article Title: Novel role for receptor dimerization in post-translational processing and turnover of the GRα

doi: 10.1038/s41598-018-32440-z

Figure Lengend Snippet: Loss of GRα dimerization, due to CpdA binding or use of hGRdim mutant, restricts hyper-phosphorylation at pS404. COS-1 cells were seeded in a 12 well plate (1 × 10 5 cell/well) and the next day transiently transfected with either hGRwt (400 ng/well) ( A ) or hGRdim (800 ng/well) ( B ). Following 24 hours incubation, cells were treated with compounds Dex, F and CpdA (10 −5 M) for 2 hours. This experiment was repeated in HepG2 cells seeded in a 12 well plate (1 × 10 5 cell/well), containing endogenous GRα ( C ). Additionally, HepG2 cells were treated with 5 μM BIO (GSK3β inhibitor) for 1 hour prior to treatment with compounds ( D ). pS404-GR levels in ( A – C ) were detected using Western blotting, which were quantified using UNSCANIT. Blots were stripped and re-probed for total GRα protein content for normalization. In the case of ( D ), total hGRα was detected and normalized to loading control GAPDH. Normalized values were then plotted and expressed as a percentage relative to solvent (EtOH), which was set at 100% and is represented by the dotted line. A representative blot from a single experiment is shown. For statistical analysis, of hGRwt ( A ) and endogenous hGRα ( C ), a one-way ANOVA followed by a Tukey’s multiple comparisons post-test (ns, P > 0.05, *P < 0.05, **P < 0.01 and ***P < 0.001) was conducted on logarithmically transformed data. An unpaired two-tailed t-test with Welch’s correction was conducted comparing hGRα protein levels in the absence or presence of BIO, following Dex, F or CpdA treatment in ( D ). Full-length blots are presented in Supplementary Fig. .

Article Snippet: African green monkey kidney ( COS-1) cells and the human liver carcinoma cell line (HepG2), containing endogenous GRα, were purchased from American Type Culture Collection (USA).

Techniques: Binding Assay, Mutagenesis, Phospho-proteomics, Transfection, Incubation, Western Blot, Control, Solvent, Transformation Assay, Two Tailed Test

Journal: Scientific Reports

Article Title: Novel role for receptor dimerization in post-translational processing and turnover of the GRα

doi: 10.1038/s41598-018-32440-z

Figure Lengend Snippet: Ligand-dependent subcellular localization of GRα modulates its co-localization with endogenous FBXW7α. COS-1 cells were seeded into a 10 cm dish (1 × 10 6 cells) and transiently transfected with either hGRwt ( A and C ) or hGRdim ( B and D ). Following 24 hours incubation, cells were re-plated and treated with solvent (EtOH), Dex, F or CpdA (10 −5 M) for 3 hours. Thereafter, cells were fixed, permeabilised, and immunofluorescence conducted, with antibodies specific for GRα and FBXW7α. Cells were then imaged using a confocal microscope. For the quantification of the subcellular localisation of ( A ) hGRwt or ( B ) hGRdim the relative fluorescence intensity (RFI) of the red (GRα) pixels was calculated for individual cells by selecting regions of interest (ROI), and plotted. In addition, the co-localisation of GRα and FBXW7α, in terms of hGRwt ( C ) or hGRdim ( D ), was determined using the weighted co-localisation coefficients, and expressed as a percentage, where the horizontal dotted line represents 100% co-localization of GRα (hGRwt or hGRdim) with FBXW7α. To compare the cytoplasmic and nuclear expression of hGRwt ( A ) or hGRdim ( B ) within a treatment group statistical analysis was conducted on logarithmically transformed data using a two-way ANOVA followed by a Bonferroni multiple comparisons post-test (ns, P > 0.05 and ***P < 0.001). In addition, comparing localization of the hGRwt ( A ) or hGRdim ( B ) of all samples to each other, a one-way ANOVA with a Tukey’s multiple comparisons post-test was conducted on logarithmically transformed data (for a, b, c and d, letters that are the same represent no significant difference between values whilst letters, which are different are significantly different from each other P < 0.05). Comparing ligand-induced co-localization of hGRwt ( C ) or hGRdim ( D ) with FBXW7α statistical analysis was conducted on logarithmically transformed data using a one-way ANOVA with a Dunnett’s multiple comparisons post-test, comparing experimental values to the solvent (EtOH) (ns, P > 0.05 and ***P < 0.001).

Article Snippet: African green monkey kidney ( COS-1) cells and the human liver carcinoma cell line (HepG2), containing endogenous GRα, were purchased from American Type Culture Collection (USA).

Techniques: Transfection, Incubation, Solvent, Immunofluorescence, Microscopy, Fluorescence, Expressing, Transformation Assay

Journal: Scientific Reports

Article Title: Novel role for receptor dimerization in post-translational processing and turnover of the GRα

doi: 10.1038/s41598-018-32440-z

Figure Lengend Snippet: Loss of GRα dimerization modulates its interaction with FBXW7α. COS-1 cells were seeded, transfected and re-plated into 8 well chambers. Following 24 hours incubation, cells were treated with solvent (EtOH), Dex, F or CpdA (10 −5 M) for 3 hours. For the co-IP ( A ) experiment, cells were lysed after compound treatment and FBXW7α was immuno-precipitated with a GRα antibody. Western blotting was conducted on Inputs to probe for GRα, FBXW7α and GAPDH and on IP to probe for FBXW7α. A representative blot of three independent experiments is shown (IP: row 1 and Inputs: rows 2–4). For quantification, the intensity of, hGRwt, FBXW7α and GAPDH were determined using the MyECL Image Software Analysis. Moreover, the hGRwt/FBXW7α interaction was normalized to hGRwt input expression and expressed as a percentage relative to EtOH. The dotted line on the graph represents the hGRwt/FBXW7α interaction in the presence of solvent (EtOH), and is set at 100%. For the PLA ( B ), following treatment cells were fixed, permeabilized and PLA conducted using specific antibodies for GRα and FBXW7α, after which cells were imaged. A representative image of individual cells from the GFP-tagged GRα and FBXW7α (B, inset below graph) experiment, is shown. In these representative images the PLA signal is observed as distinct red spots and the cell’s nucleus is depicted by the blue DAPI stain. For quantification of the GFP-tagged GRα and FBXW7α interaction ( B ), the PLA signal (dots/cell) was quantified using the IMAGEJ Software and normalized to the GRα concentration (i.e. GFP-signal (RFI)), which was determined using the ZENN 2012 Software Analysis, and plotted. To compare GRα hGRwt/FBXW7α interaction in response to the test compounds relative to solvent (EtOH) using Co-IP ( A ), statistical analysis was conducted on logarithmically transformed data using a one-way ANOVA followed by a Dunnett’s multiple comparisons post-test (ns, P > 0.05, *P < 0.05 and ***P < 0.001). PLA analysis of GFP-hGRwt or GFP-hGRdim interacting with FBXW7α in response to the test compounds ( B ) were compared using one-way ANOVA followed by a Tukey’s multiple comparisons post-test on logarithmically transformed data (ns, P > 0.05 and ***P < 0.001). Full-length blots are presented in Supplementary Fig. .

Article Snippet: African green monkey kidney ( COS-1) cells and the human liver carcinoma cell line (HepG2), containing endogenous GRα, were purchased from American Type Culture Collection (USA).

Techniques: Transfection, Incubation, Solvent, Co-Immunoprecipitation Assay, Western Blot, Software, Expressing, Staining, Concentration Assay, Transformation Assay

Journal: Scientific Reports

Article Title: Novel role for receptor dimerization in post-translational processing and turnover of the GRα

doi: 10.1038/s41598-018-32440-z

Figure Lengend Snippet: Co-treatment with CpdA partially restores hGRwt protein levels. COS-1 cells were seeded into a 24 well plate (5 × 10 4 cells/well) and transiently transfected with hGRwt. Following 24 hours incubation, cells were treated individually with solvent (EtOH), Dex (1 μM or 10 μM) or CpdA (10 μM) or with CpdA (10 μM) in combination with Dex (1 μM or 10 μM) for 24 hours. hGRwt protein expression was confirmed by Western blotting where GAPDH was probed to ensure equal protein loading. Western blot shown (inset) is representative of four independent experiments. For quantification, the intensity of the hGRwt and GAPDH bands were determined using My ECL Image Analysis software and hGRwt levels were then normalized to GAPDH expression. hGRwt expression is expressed as a percentage (average ± SEM) of hGRwt expression in presence of the solvent (EtOH), which was set at 100% (dotted line). To determine the effect of each treatment condition on hGRwt protein expression relative to the solvent (EtOH), statistical analysis was conducted on logarithmically transformed data using a one-way ANOVA with a Dunnett’s multiple comparisons post-test (ns, P > 0.05 and ***P < 0.001). To assess the ability of CpdA to preserve hGRwt expression in the presence of Dex an unpaired two-tailed t-test with Welch’s correction was conducted comparing hGRwt expression following Dex treatment (either 1 μM or 10 μM) alone to Dex treatment in combination with CpdA (10 μM) ( # P < 0.05, ## P < 0.01). Full-length blots are presented in Supplementary Fig. .

Article Snippet: African green monkey kidney ( COS-1) cells and the human liver carcinoma cell line (HepG2), containing endogenous GRα, were purchased from American Type Culture Collection (USA).

Techniques: Transfection, Incubation, Solvent, Expressing, Western Blot, Software, Transformation Assay, Two Tailed Test