Journal: iScience

Article Title: Acetylation-induced proteasomal degradation of the activated glucocorticoid receptor limits hormonal signaling

doi: 10.1016/j.isci.2024.108943

Figure Lengend Snippet: GR acetylation attenuates chromatin binding and protein stability upon prolonged GC exposure (A) ChIP-qPCR showing GILZ (Gene name: TSC22D3 ) and FKBP5 GRE occupancy of GR in HepG2 GR −/− + WT-GR and HepG2 GR −/− + K154R cells treated with vehicle or 1 μM Dex for 24 h. Scheme above shows qPCR amplicon positions for GILZ (purple) and FKBP5 (orange) GRE with respect to GR ChIP-seq peaks in HepG2 cells from. Data are shown as percentage of input. IgG served as a control for ChIP background. (B) Western blot analysis of CHX chase experiment. HepG2 GR −/− cells transiently overexpressing Ty1-tagged WT or K154R-GR were treated with 1 μM Dex for 2 h followed by 20 ng/mL CHX for the indicated durations. Quantification shows GR/ACTB ratios normalized to respective 0 h CHX control. (C) CHX chase experiment as in (B) performed in HepG2 cells treated with 1 μM Dex+DMSO or 1 μM Dex+20 μM Ex527. Quantification shows GR/TUBA ratios normalized to respective 0 h CHX controls. (D) Western blot analysis of polyubiquitinated FLAG-GR immunoprecipitated from HEK239 GR −/− transiently expressing FLAG-tagged WT- or K154R-GR along with HA-UBB, treated with 1 μM Dex with or without 1 μM MG132 for 24 h. Quantification represents ratio of HA-UBB/GR signals from lanes 2 and 4 normalized to WT-Dex+MG132 (lane 2) sample. Data represent mean of n = 3 except for (A) where n = 4 independent experiments. Error bars = S.E.M. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, n.s = non-significant; (B, C): One way ANOVA-Fischer’s LSD test; (A) Student’s paired t-test and (D) Welch’s t-test. See also Figure S5 A.

Article Snippet: 15-20 μg chromatin was incubated overnight at 4°C with antibodies coupled to protein G dynabeads (Invitrogen) blocked with BSA and herring sperm DNA: for GR ChIP a cocktail of 5 μl anti-GR D6H2L-XP rabbit monoclonal (Cell signaling Technology), 1 μg anti-GR H-300 rabbit polyclonal (Santa Cruz) and 5 μg of Ty1 (Diagenode) monoclonal antibodies was used and mouse/rabbit IgG (Sigma-Aldrich/Cell Signaling Technology) served as control.

Techniques: Binding Assay, Amplification, ChIP-sequencing, Western Blot, Immunoprecipitation, Expressing

Journal: iScience

Article Title: Acetylation-induced proteasomal degradation of the activated glucocorticoid receptor limits hormonal signaling

doi: 10.1016/j.isci.2024.108943

Figure Lengend Snippet:

Article Snippet: 15-20 μg chromatin was incubated overnight at 4°C with antibodies coupled to protein G dynabeads (Invitrogen) blocked with BSA and herring sperm DNA: for GR ChIP a cocktail of 5 μl anti-GR D6H2L-XP rabbit monoclonal (Cell signaling Technology), 1 μg anti-GR H-300 rabbit polyclonal (Santa Cruz) and 5 μg of Ty1 (Diagenode) monoclonal antibodies was used and mouse/rabbit IgG (Sigma-Aldrich/Cell Signaling Technology) served as control.

Techniques: Generated, Recombinant, Cell Culture, Synthesized, Luciferase, Isolation, Multiplex Assay, Software

Journal: British Journal of Cancer

Article Title: In breast cancer subtypes steroid sulfatase (STS) is associated with less aggressive tumour characteristics

doi: 10.1038/s41416-018-0034-9

Figure Lengend Snippet: Breast cancer subtype and marker expression

Article Snippet: Immunohistochemistry (IHC) for AR, GR, CYP19 (aromatase), 17βHSD2, STS, FOXA1, and ERβ was performed, as previously described., – In brief, the following primary antibodies and conditions were employed; (Ki67 (MIB-1) DAKO 1:100; AR (AR441)DAKO 1:50; GR, (D6H2L)Cell Signalling technologies 1:400; AROM (677), Novartis, 1:500; 17βHSD2, Proteintech, 1:200; STS, (KW1049) Kyowa medix, 1:100; FOXA1 (3C1) ABCAM 1:200, ERβ1 1:1000 (Genetex, 14C8)).

Techniques: Marker

Journal: British Journal of Cancer

Article Title: In breast cancer subtypes steroid sulfatase (STS) is associated with less aggressive tumour characteristics

doi: 10.1038/s41416-018-0034-9

Figure Lengend Snippet: Regression analysis

Article Snippet: Immunohistochemistry (IHC) for AR, GR, CYP19 (aromatase), 17βHSD2, STS, FOXA1, and ERβ was performed, as previously described., – In brief, the following primary antibodies and conditions were employed; (Ki67 (MIB-1) DAKO 1:100; AR (AR441)DAKO 1:50; GR, (D6H2L)Cell Signalling technologies 1:400; AROM (677), Novartis, 1:500; 17βHSD2, Proteintech, 1:200; STS, (KW1049) Kyowa medix, 1:100; FOXA1 (3C1) ABCAM 1:200, ERβ1 1:1000 (Genetex, 14C8)).

Techniques:

Journal: British Journal of Cancer

Article Title: In breast cancer subtypes steroid sulfatase (STS) is associated with less aggressive tumour characteristics

doi: 10.1038/s41416-018-0034-9

Figure Lengend Snippet: The impact of steroidogenic proteins on overall survival. We detected an effect of STS ( a ), 17βHSD2 ( b ), and GR ( c ) expression on overall survival rates with high levels of STS being associated with longer survival while high levels of 17βHSD2 and GR were associated with shorter survival. Survival analysis examining the interactions of STS expression with breast cancer subtype ( d ) and endocrine therapy ( e ) revealed that the survival benefit associated with STS expression was not confined to one breast cancer subtype or related to a specific endocrine intervention

Article Snippet: Immunohistochemistry (IHC) for AR, GR, CYP19 (aromatase), 17βHSD2, STS, FOXA1, and ERβ was performed, as previously described., – In brief, the following primary antibodies and conditions were employed; (Ki67 (MIB-1) DAKO 1:100; AR (AR441)DAKO 1:50; GR, (D6H2L)Cell Signalling technologies 1:400; AROM (677), Novartis, 1:500; 17βHSD2, Proteintech, 1:200; STS, (KW1049) Kyowa medix, 1:100; FOXA1 (3C1) ABCAM 1:200, ERβ1 1:1000 (Genetex, 14C8)).

Techniques: Expressing