Journal: Cell death discovery

Article Title: Exploring the potential of selective FKBP51 inhibitors on melanoma: an investigation of their in vitro and in vivo effects.

doi: 10.1038/s41420-025-02430-y

Figure Lengend Snippet: Fig. 1 SAFit enhances melanoma cell death. a Dose response assay of SAFit 1 and 2 on Doxo-induced cell death. A375 melanoma cells were cultured in the presence or absence of 3 μM Doxo and/or SAFit 1 (4, 20 and 40 nM) and SAFit 2 (6, 30 and 60 nM). After a 48 h incubation, cell death was assessed by measuring the proportion of hypodiploid cells by flow cytometry. b (Left) Western blot assay showing exogenous levels of FKBP51, β-Actin was used as loading control. Full length western blots are shown as Supplemental Material. (Right) Graphical representation of cell death values (N = 4) from cultures of Flag-FKBP51 and EV incubated with 3 μM Doxo and/or 20 nM SAFit 1. After a 48 h incubation, cells were harvested and analysed by PI incorporation and flow cytometry. Representative flow cytometry histograms of PI incorporation are shown below. c SAFit improves dacarbazine (DTIC)-induced cell death. SAN melanoma cells were cultured in the presence or absence of 27 μM DTIC and with or without 100 nM SAFit 1 or 100 nM rapamycin. After 48 h incubation cells were harvested and analysed by flow cytometry. The graph shows cell death values obtained from independent experiments (N = 3). Representative flow cytometry histograms of PI incorporation are shown below.

Article Snippet: Bcl-2 (N-19, rabbit polyclonal, Santa Cruz Biotechnology), XIAP (2F1, mouse monoclonal, Stressgen Biotechnologies, BC, Canada), FKBP51 (NB100-68240, Novus Biological), Cyclin D1 (92G2, rabbit polyclonal, Cell Signaling Technology).

Techniques: Cell Culture, Incubation, Cytometry, Western Blot, Control

Journal: Cell death discovery

Article Title: Exploring the potential of selective FKBP51 inhibitors on melanoma: an investigation of their in vitro and in vivo effects.

doi: 10.1038/s41420-025-02430-y

Figure Lengend Snippet: Fig. 2 SAFits counteract NF-κB/Rel activation. a Electrophoretic mobility shift assay (EMSA) of nuclear extracts obtained from SAN melanoma cells cultured with 3 μM Doxo and/or SAFit1 20 nM or SAFit2 30 nM, for 3 and 5 h. A competition assay with the same cold oligo or an unrelated (NF-AT) oligo suggests the specificity of NF-κB bands. Full gels are shown as Supplemental Material. b Relative normalized expression values of BCL-2 and XIAP mRNA levels in SAN melanoma cells incubated for 24 h in the presence or absence of 20 nM SAFit1 or 30 nM SAFit2. Relative quantitation of the transcript was performed using co-amplified β-Actin as an internal control for normalization. (Lower), Western blot of protein extracted from the same cells for Bcl-2 and XIAP assay. Full gels are shown as Supplemental Material. c BCL-2 and FKBP51 mRNA levels in FKBP51-knocked down A375 melanoma cells (Sh FKBP51 RNA), transfected or not with Flag-FKBP51. (Lower), Western blot of protein extracted from the same cells for Bcl-2, FKBP51 and FLAG-FKBP51 assay. Full gels are shown as Supplemental Material.

Article Snippet: Bcl-2 (N-19, rabbit polyclonal, Santa Cruz Biotechnology), XIAP (2F1, mouse monoclonal, Stressgen Biotechnologies, BC, Canada), FKBP51 (NB100-68240, Novus Biological), Cyclin D1 (92G2, rabbit polyclonal, Cell Signaling Technology).

Techniques: Activation Assay, Electrophoretic Mobility Shift Assay, Cell Culture, Competitive Binding Assay, Expressing, Incubation, Quantitation Assay, Control, Western Blot, Transfection

Journal: Molecular Cancer Therapeutics

Article Title: Cell Intrinsic Role of COX-2 in Pancreatic Cancer Development

doi: 10.1158/1535-7163.mct-12-0342

Figure Lengend Snippet: Figure 7. Decreased FKBP5 negative feedback leads to enhanced AKT activation in Pdx1-Creþ;K-rasG12D/þ;Ptenlox/þ; Cox-2lox/lox mice. A, FKBP5 mRNA expression in PDACs of Pdx1-Creþ; K-rasG12D/þ;Ptenlox/þ;Cox-2lox/lox

Article Snippet: The following primary antibodies were used: phospho-AKT(Ser473) (Cell Signaling; 1:50), Cytokeratin 19 (ab15463, Abcam; 1:100), COX-2 (SP21; Thermo Scientific, ready-to-use),GRP78(11587-1AP,ProteinTechGroup; 1:50), and FKBP5 (14155-1-AP, ProteinTech Group, 1:50).

Techniques: Activation Assay, Expressing

Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: Molecular Pathways Leading to Induction of Cell Death and Anti-Proliferative Properties by Tacrolimus and mTOR Inhibitors in Liver Cancer Cells.

doi: 10.33594/000000230

Figure Lengend Snippet: Fig. 7. FKBP12 and FKBP51 protein expressions in Tacrolimus-, Sirolimus- and Everolimus-treated HepG2 (A and B, respectively) and Huh7 (C and D, respectively) cells. Treatments were administered at different concentrations (0, 10 nM, 10 µM, and 100 µM). The protein expression of FKBP12 and FKBP51 was evaluated by Western‐blot analysis as described in Material and Methods. Results are expressed as mean ± SEM, and blots are representative of four to six independent experiments. *p ≤ 0.05 between control and immunosuppressant‐treated cells. The groups with different letters (a, b, c or d) were significantly different (p ≤ 0.05).

Article Snippet: KG Navarro-Villarán et al.: Role of Immunosuppressant and FK506-Binding Protein Complex in Liver Cancer and Ser15P-p53 (#9284) obtained from Cell Signaling Technology (Danvers, Massachusetts, USA); LC3 (PM036) purchased from MBL International (Woburn, Massachusetts, USA); GADD153 (C/EBP homologous protein or CHOP) (sc-575), Beclin (sc-48341), p21 (sc-397) and p53 (sc-6243) obtained from Santa Cruz Biotechnology (Dallas, Texas, USA); Thr172P-Cdk4 (PA5-64482) obtained from ThermoFisher (Waltham, Massachusetts, USA); FKBP12 (Ref NB300-508) and FKBP38 (Ref NBP1-77909) obtained from Novus Biologicals (Centennial, Colorado, USA); and FKBP51 (Ref MAB4094) and FKBP52 (Ref MAB4095) obtained from R&D Systems (Minneapolis, Minnesota, USA).

Techniques: Expressing, Western Blot, Control

Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: Molecular Pathways Leading to Induction of Cell Death and Anti-Proliferative Properties by Tacrolimus and mTOR Inhibitors in Liver Cancer Cells.

doi: 10.33594/000000230

Figure Lengend Snippet: Fig. 9. Impact of FKBP51 downregulation on BrdU incorporation (A) and caspase-3 activity (B) in Tacrolimus-, Sirolimus- and Everolimus-treated HepG2 cells. The downregulation of FKBP51 was carried using siRNA technologies. Cell proliferation and apoptosis were determined using commercial BrdU incorporation and caspase‐3 activity assays respectively as described in Material and Methods. Results are expressed as mean ± SEM of six independent experiments. *p ≤ 0.05 and **p ≤ 0.01 between control and immunosuppressant‐ treated cells. The groups with different letters (a, b, c, d, e or f) were significantly different (p ≤ 0.05).

Article Snippet: KG Navarro-Villarán et al.: Role of Immunosuppressant and FK506-Binding Protein Complex in Liver Cancer and Ser15P-p53 (#9284) obtained from Cell Signaling Technology (Danvers, Massachusetts, USA); LC3 (PM036) purchased from MBL International (Woburn, Massachusetts, USA); GADD153 (C/EBP homologous protein or CHOP) (sc-575), Beclin (sc-48341), p21 (sc-397) and p53 (sc-6243) obtained from Santa Cruz Biotechnology (Dallas, Texas, USA); Thr172P-Cdk4 (PA5-64482) obtained from ThermoFisher (Waltham, Massachusetts, USA); FKBP12 (Ref NB300-508) and FKBP38 (Ref NBP1-77909) obtained from Novus Biologicals (Centennial, Colorado, USA); and FKBP51 (Ref MAB4094) and FKBP52 (Ref MAB4095) obtained from R&D Systems (Minneapolis, Minnesota, USA).

Techniques: BrdU Incorporation Assay, Activity Assay, Control

Journal: bioRxiv

Article Title: Epigenetic derepression of FKBP5 by aging and stress contributes to NF-ĸB-driven inflammation and cardiovascular risk

doi: 10.1101/484709

Figure Lengend Snippet: (A) Methylation decreases at selected FKBP5 CpGs along the human lifespan (GTP: β age = -0.0045, SE = 0.0008, p = 8 x 10 -8 ; KORA: β age = -0.0055, SE = 0.0005, p < 2 x 10 -16 ; MPIP: β age = -0.0064, SE = 0.0012, p = 7 x 10 -8 ; total n = 2,523). (B) Depressive phenotypes are associated with accelerated age-related FKBP5 demethylation (total n = 2,249, meta-analysis interaction p = 2.6 x 10 -2 , heterogeneity p = 2.7 x 10 -1 ). Statistics per cohort: GTP: interaction p = 1.9 x 10 -2 , β age for moderate/severe depression = -0.0075 (SE = 0.0014) vs. β age for no/mild depression = -0.0032 (SE = 0.0011); KORA: interaction p = 6.3 x 10 -1 , β age for higher levels of depression = -0.0063 (SE = 0.0011) vs. β age for lower levels of depression = -0.0047 (SE = 0.0007); MPIP: interaction p = 1.9 x 10 -1 , β age for depressed = -0.0077 (SE = 0.0015) vs. β age for non-depressed = -0.0044 (SE = 0.0019). (C) Early life separation is associated with demethylation of the age-related FKBP5 CpGs in the HBCS (βseparation = - 0.0932, SE = 0.0343, p = 7.4 x 10 -3 , mean DNA methylation difference = 1.4%). All coefficients and p values are derived from linear regression models using M-values for DNA methylation and after correcting for potential confounders (see Methods). (D) In vitro aging and exposure to the stress hormone (glucocorticoid) receptor agonist dexamethasone (DEX) additively decrease methylation at the age-related FKBP5 CpGs in the IMR-90 fibroblast model of replicative senescence (F 1,6 = 6.3, interaction p = 4.6 x 10 -2 , n = 4 replicates per age group). Statistical comparisons were performed with two-way mixed-design ANOVA (according to experimental design), using replicative age as the between-subject and DEX treatment as the within-subject factor. Statistically significant effects were followed with Bonferroni-corrected pairwise comparisons, shown as follows: * p < 5 x 10 -2 , statistically significant pairwise comparisons for young vs. old replicative age; # p < 5 x 10 -2 , statistically significant pairwise comparison for vehicle vs. DEX-treated old cells. Error bars depict the standard error around the group mean. The y axes in panels (A), (B), and (C) depict the average DNA methylation levels of the two age-related FKBP5 CpGs (cg20813374 and cg00130530), after adjustment for the respective covariates for each cohort; for a more intuitive visualization, selected panels are also depicted as % DNA methylation (Beta-values in Supplementary Fig. 1). The y axis in panel (D) depicts the average % DNA methylation (Beta-values) of the two FKBP5 CpGs. GTP, Grady Trauma Project; HBCS, Helsinki Birth Cohort Study; KORA, Cooperative Health Research in the Region of Augsburg F4 community study; MPIP, Max Planck Institute of Psychiatry depression case/control study.

Article Snippet: The following primary antibodies were used: FLAG (1:7,000, Rockland, 600-401-383), FKBP5/FKBP51 (1:1,000, Bethyl, A301-430A; 1:1000, Cell Signaling, #8245), NF-κB/p65 (1:1000, Cell Signaling, #8242), IKKα (1:1000, Cell Signaling, #2682), pIKKα S176 (1:1000, Cell Signaling, #2078), NIK (1:1000, Cell Signaling, #4994), and Actin (1:5,000, Santa Cruz, sc-1616).

Techniques: Methylation, DNA Methylation Assay, Derivative Assay, In Vitro

Journal: bioRxiv

Article Title: Epigenetic derepression of FKBP5 by aging and stress contributes to NF-ĸB-driven inflammation and cardiovascular risk

doi: 10.1101/484709

Figure Lengend Snippet: Complementary figure demonstrating how % FKBP5 DNA methylation levels are associated with aging, early life separation, and history of myocardial infarction. The y axis in all panels depicts the average % DNA methylation (Beta-values) of the two age- and stress-related FKBP5 CpGs (cg20813374 and cg00130530). Further details and statistics are provided in main and . GTP, Grady Trauma Project; HBCS, Helsinki Birth Cohort Study; KORA, Cooperative Health Research in the Region of Augsburg F4 community study; MI, myocardial infarction; MPIP, Max Planck Institute of Psychiatry depression case/control study.

Article Snippet: The following primary antibodies were used: FLAG (1:7,000, Rockland, 600-401-383), FKBP5/FKBP51 (1:1,000, Bethyl, A301-430A; 1:1000, Cell Signaling, #8245), NF-κB/p65 (1:1000, Cell Signaling, #8242), IKKα (1:1000, Cell Signaling, #2682), pIKKα S176 (1:1000, Cell Signaling, #2078), NIK (1:1000, Cell Signaling, #4994), and Actin (1:5,000, Santa Cruz, sc-1616).

Techniques: DNA Methylation Assay

Journal: bioRxiv

Article Title: Epigenetic derepression of FKBP5 by aging and stress contributes to NF-ĸB-driven inflammation and cardiovascular risk

doi: 10.1101/484709

Figure Lengend Snippet: Validation of the 450K-identified age-regulated FKBP5 CpGs (cg20813374 and cg00130530) using targeted bisulfite sequencing with the Illumina MiSeq in a sample of female subjects (n = 77, β age = -0.0074, SE = 0.0031, p = 1.9 x 10 -2 ). The y axis depicts the average % DNA methylation (Beta-values) of the two FKBP5 CpGs. Reported statistics are after correcting for potential confounders (see Methods).

Article Snippet: The following primary antibodies were used: FLAG (1:7,000, Rockland, 600-401-383), FKBP5/FKBP51 (1:1,000, Bethyl, A301-430A; 1:1000, Cell Signaling, #8245), NF-κB/p65 (1:1000, Cell Signaling, #8242), IKKα (1:1000, Cell Signaling, #2682), pIKKα S176 (1:1000, Cell Signaling, #2078), NIK (1:1000, Cell Signaling, #4994), and Actin (1:5,000, Santa Cruz, sc-1616).

Techniques: Methylation Sequencing, DNA Methylation Assay

Journal: bioRxiv

Article Title: Epigenetic derepression of FKBP5 by aging and stress contributes to NF-ĸB-driven inflammation and cardiovascular risk

doi: 10.1101/484709

Figure Lengend Snippet: Visual depiction of integrative analysis of chromatin states using ChromHMM (ref 52) within immune cell types, as well as in the IMR-90 fibroblasts that were used as a model of human replicative senescence. Across these cell types, the age- and stress-related FKBP5 CpGs are commonly mapped to an enhancer or flanking active TSS (further details in Supplementary Table 3).

Article Snippet: The following primary antibodies were used: FLAG (1:7,000, Rockland, 600-401-383), FKBP5/FKBP51 (1:1,000, Bethyl, A301-430A; 1:1000, Cell Signaling, #8245), NF-κB/p65 (1:1000, Cell Signaling, #8242), IKKα (1:1000, Cell Signaling, #2682), pIKKα S176 (1:1000, Cell Signaling, #2078), NIK (1:1000, Cell Signaling, #4994), and Actin (1:5,000, Santa Cruz, sc-1616).

Techniques:

Journal: bioRxiv

Article Title: Epigenetic derepression of FKBP5 by aging and stress contributes to NF-ĸB-driven inflammation and cardiovascular risk

doi: 10.1101/484709

Figure Lengend Snippet: Functional annotation of the FKBP5 locus at and around the age-related FKBP5 CpGs, using the Roadmap Epigenome Browser and the “Mobilized CD34 Primary cells” track as a proxy for peripheral immune cells. The two CpGs (cg20813374 and cg00130530) are respectively located at positions 35657180 and 35657202 of chromosome 6 (exact location indicated by dotted line) and, as shown, exhibit intermediate methylation levels and colocalize with H3K4me1 and H3K27me3 signatures. This landscape is most consistent with a poised enhancer (ref 53).

Article Snippet: The following primary antibodies were used: FLAG (1:7,000, Rockland, 600-401-383), FKBP5/FKBP51 (1:1,000, Bethyl, A301-430A; 1:1000, Cell Signaling, #8245), NF-κB/p65 (1:1000, Cell Signaling, #8242), IKKα (1:1000, Cell Signaling, #2682), pIKKα S176 (1:1000, Cell Signaling, #2078), NIK (1:1000, Cell Signaling, #4994), and Actin (1:5,000, Santa Cruz, sc-1616).

Techniques: Functional Assay, Methylation

Journal: bioRxiv

Article Title: Epigenetic derepression of FKBP5 by aging and stress contributes to NF-ĸB-driven inflammation and cardiovascular risk

doi: 10.1101/484709

Figure Lengend Snippet: (A) FKBP5 expression levels are negatively associated with average methylation of the age-related sites (β = -0.3835, SE = 0.1585, p = 1.6 x 10 -2 ). (B and C) The cortisol- FKBP5 relationship is stronger at lower methylation levels of the age-related FKBP5 CpGs: interaction p = 1.4 x 10 -3 , β cortisol for lower methylation = 0.0299 (SE = 0.0044) vs. β cortisol for higher methylation = 0.0069 (SE = 0.0039). The cortisol- FKBP5 relationship is stronger in older ages: interaction p = 2.4 x 10 -5 , β cortisol for older subjects = 0.0376 (SE = 0.0050) vs. β cortisol for younger subjects = 0.0075 (SE = 0.0035). (D) Higher levels of depressive symptoms are associated with stronger cortisol- FKBP5 relationship in subjects with higher levels of childhood trauma (cortisol-depression interaction p = 7.3 x 10 -5 ) but not in subjects with lower levels of childhood trauma (cortisol-depression interaction p = 1.4 x 10 -1 ) as defined with the Childhood Trauma Questionnaire (CTQ). Panel (A) depicts the average % methylation levels (Beta-values) of the two age-related FKBP5 CpGs (cg20813374 and cg00130530).

Article Snippet: The following primary antibodies were used: FLAG (1:7,000, Rockland, 600-401-383), FKBP5/FKBP51 (1:1,000, Bethyl, A301-430A; 1:1000, Cell Signaling, #8245), NF-κB/p65 (1:1000, Cell Signaling, #8242), IKKα (1:1000, Cell Signaling, #2682), pIKKα S176 (1:1000, Cell Signaling, #2078), NIK (1:1000, Cell Signaling, #4994), and Actin (1:5,000, Santa Cruz, sc-1616).

Techniques: Expressing, Methylation

Journal: bioRxiv

Article Title: Epigenetic derepression of FKBP5 by aging and stress contributes to NF-ĸB-driven inflammation and cardiovascular risk

doi: 10.1101/484709

Figure Lengend Snippet: Methylation levels at the age- and stress-related FKBP5 CpGs (cg20813374 and cg001305030) inversely correlate with FKBP5 mRNA levels in breast tissue samples of control female subjects (n = 84). Publicly available data were analyzed from the Cancer Genome Atlas Wanderer ( http://maplab.imppc.org/wanderer/ ; ref 54). The x axis depicts the average % DNA methylation (Beta-values) of the CpGs. The y axis depicts log2-transformed normalized RSEM RNAseq-measured FKBP5 expression.

Article Snippet: The following primary antibodies were used: FLAG (1:7,000, Rockland, 600-401-383), FKBP5/FKBP51 (1:1,000, Bethyl, A301-430A; 1:1000, Cell Signaling, #8245), NF-κB/p65 (1:1000, Cell Signaling, #8242), IKKα (1:1000, Cell Signaling, #2682), pIKKα S176 (1:1000, Cell Signaling, #2078), NIK (1:1000, Cell Signaling, #4994), and Actin (1:5,000, Santa Cruz, sc-1616).

Techniques: Methylation, DNA Methylation Assay, Transformation Assay, Expressing

Journal: bioRxiv

Article Title: Epigenetic derepression of FKBP5 by aging and stress contributes to NF-ĸB-driven inflammation and cardiovascular risk

doi: 10.1101/484709

Figure Lengend Snippet: (A) FKBP5 -related genes in peripheral blood show enrichment for inflammation-related genes and NF-κB gene targets. Disease association and transcription factor target analyses were performed using genome-wide gene expression data in the Grady Trauma Project cohort (GTP; n = 355). The number of genes for each analysis is shown in parentheses. Statistical details are provided in Supplementary Tables 5-9. (B) Western blotting confirming FKBP5 overexpression in Jurkat T cells transfected with FKBP51-FLAG vs. cells transfected with the control vector. (C) FKBP5 overexpression nearly doubles IL-8 secretion by Jurkat T cells stimulated overnight with 25 ng/ml of Phorbol-12-myristate-13-acetate and 375 ng/ml of ionomycin (PMA/I). The bar graph depicts IL-8 secretion in stimulated cell supernatants measured with ELISA from two independent experiments (t = 8.8, p = 4.4 x 10 -7 , n = 8 per condition). For each experiment, fold ratios of IL-8 secretion were calculated relative to stimulated cells expressing the control vector. IL-8 was not detectable in non-stimulated cells (not shown). (D) FKBP5 overexpression increases NF-κB activity in stimulated Jurkat T cells. The bar graph depicts NF-κB reporter activity in stimulated cells measured with dual-luciferase reporter assays from three independent experiments (t = 3.2, p = 5.5 x 10 -3 , n = 9 per condition). For each experiment, fold ratios of NF-κB activity were calculated relative to non-stimulated cells expressing the control vector. (E) FKBP5 expression changes are associated with extensive alterations in the NF-κB co-expression network in the GTP (n = 355). The circles depict genes encoding molecular partners of the NF-κB pathway. Continuous lines (edges) represent positive and dotted lines negative pairwise correlations corrected for expression levels of all other genes in the pathway (details in Methods). Edge widths are proportional to the absolute value of the respective correlation coefficient. The gene pair with the most robust difference in correlation between the two groups ( CHUK-MAP3K14 ) is highlighted in orange. Statistical details for all gene pairs are provided in Supplementary Table 10. Error bars depict the standard error around the group mean. ** p < 10 -2 ; *** p < 10 -3 .

Article Snippet: The following primary antibodies were used: FLAG (1:7,000, Rockland, 600-401-383), FKBP5/FKBP51 (1:1,000, Bethyl, A301-430A; 1:1000, Cell Signaling, #8245), NF-κB/p65 (1:1000, Cell Signaling, #8242), IKKα (1:1000, Cell Signaling, #2682), pIKKα S176 (1:1000, Cell Signaling, #2078), NIK (1:1000, Cell Signaling, #4994), and Actin (1:5,000, Santa Cruz, sc-1616).

Techniques: Genome Wide, Expressing, Western Blot, Over Expression, Transfection, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Activity Assay, Luciferase

Journal: bioRxiv

Article Title: Epigenetic derepression of FKBP5 by aging and stress contributes to NF-ĸB-driven inflammation and cardiovascular risk

doi: 10.1101/484709

Figure Lengend Snippet: Association of FKBP5 expression levels with the granulocyte to lymphocyte ratio (n = 330), an inflammation marker linked with heightened cardiovascular risk and mortality. Reported statistics and depicted residuals (on the x axis) are after correcting for covariates (see Methods).

Article Snippet: The following primary antibodies were used: FLAG (1:7,000, Rockland, 600-401-383), FKBP5/FKBP51 (1:1,000, Bethyl, A301-430A; 1:1000, Cell Signaling, #8245), NF-κB/p65 (1:1000, Cell Signaling, #8242), IKKα (1:1000, Cell Signaling, #2682), pIKKα S176 (1:1000, Cell Signaling, #2078), NIK (1:1000, Cell Signaling, #4994), and Actin (1:5,000, Santa Cruz, sc-1616).

Techniques: Expressing, Marker

Journal: bioRxiv

Article Title: Epigenetic derepression of FKBP5 by aging and stress contributes to NF-ĸB-driven inflammation and cardiovascular risk

doi: 10.1101/484709

Figure Lengend Snippet: (A) Immunoprecipitation (IP) for either FKBP5 or NIK followed by Western blotting in lysates from Jurkat cells or peripheral blood monocytes (PBMC) treated for 24 hours with the stress hormone (glucocorticoid) receptor agonist dexamethasone (DEX, 100nM), which robustly induces FKBP5 expression, and/or selective FKBP5 antagonists (SAFit1, 100nM). IgG: control IP without primary antibody. (B) Quantifications of respective IPs showing DEX-induced increase in FKBP5-NIK-IKKα binding that is prevented by concomitant treatment with SAFit1 (Jurkat: FKBP5 to NIK binding, DEX x SAFit1 F 1,8 = 9.3, interaction p = 1.6 x 10 -2 ; IKKα to FKBP5 binding, DEX x SAFit1 F 1,8 = 4.7, interaction p = 6.2 x 10 -2 ; IKKα to NIK binding, DEX x SAFit1 F 1,8 = 5.8, interaction p = 4.3 x 10 -2 . PBMC: FKBP5 to NIK binding, DEX x SAFit1 F 1,8 = 5.7, interaction p = 4.4 x 10 -2 ; IKKα to FKBP5 binding, DEX x SAFit1 F 1,8 = 11.2, interaction p = 1 x 10 -2 ; IKKα to NIK binding, DEX x SAFit1 F 1,8 = 3.9, interaction p = 8.4 x 10 -2 . n = 3 biological replicates per condition). (C) Western blotting of Jurkat cell and PBMC lysates (n = 3 replicates per condition) showing increase in the functional phosphorylation of IKKα at serine 176 (pIKKα) by 24-hour treatment with 100nM DEX, which is prevented by 24-hour treatment with 100nM SAFit1 (Jurkat: DEX x SAFit1 F 1,8 = 12.9, interaction p = 7 x 10 -3 ; PBMC: DEX x SAFit1 F 1,8 = 0.6, interaction p = 4.6 x 10 -1 ). (D) Similar effects are observed when Jurkat cells are transfected with an expression construct encoding FKBP5 and treated for 24 hours with 100nM SAFit1 (ect. FKBP5 x SAFit1 F 1,12 = 6.6, interaction p = 2.5 x 10 -2 , n = 4 replicates per condition). (E) FKBP5 overexpression increases NF-κB activity in Jurkat cells stimulated overnight with 25 ng/ml of Phorbol-12-myristate-13-acetate and 375 ng/ml of ionomycin, and this increase is prevented by concomitant treatment with 100nM SAFit1 for 24 hours (ect. FKBP5 x SAFit1 F 1,32 = 4.5, interaction p = 4.2 x 10 -2 , n = 9 replicates per condition). NF-κB reporter activity was measured with dual-luciferase reporter assays in three independent experiments. (F) Scheme summarizing the results from protein-protein binding and reporter gene experiments. All data are shown as fold changes compared to the control-vector vehicle-treated cells. All statistical comparisons were performed with two-way ANOVA, using either DEX treatment or FKBP5 overexpression as the first factor and SAFit1 treatment as the second factor. Statistically significant effects were followed with Bonferroni-corrected pairwise comparisons, shown as follows:* p < 5 x 10 -2 , ** p < 10 -2 , *** p < 10 -3 , statistically significant pairwise comparisons for control vs. DEX or ectopic FKBP5 ; # p < 5 x 10 -2 , ## p < 10 -2 , ### p < 10 -3 , significant pairwise comparisons for vehicle vs. SAFit1 treatment (shown only for significant interaction terms from two-way ANOVAs). Error bars depict the standard error around the group mean.

Article Snippet: The following primary antibodies were used: FLAG (1:7,000, Rockland, 600-401-383), FKBP5/FKBP51 (1:1,000, Bethyl, A301-430A; 1:1000, Cell Signaling, #8245), NF-κB/p65 (1:1000, Cell Signaling, #8242), IKKα (1:1000, Cell Signaling, #2682), pIKKα S176 (1:1000, Cell Signaling, #2078), NIK (1:1000, Cell Signaling, #4994), and Actin (1:5,000, Santa Cruz, sc-1616).

Techniques: Immunoprecipitation, Western Blot, Expressing, Binding Assay, Functional Assay, Transfection, Construct, Over Expression, Activity Assay, Luciferase, Protein Binding, Plasmid Preparation

Journal: bioRxiv

Article Title: Epigenetic derepression of FKBP5 by aging and stress contributes to NF-ĸB-driven inflammation and cardiovascular risk

doi: 10.1101/484709

Figure Lengend Snippet: The effect of the stress hormone (glucocorticoid) receptor agonist dexamethasone (DEX) on functional phosphorylation of IKKα at serine 176 (pIKKα) is abolished in Jurkat cells lacking FKBP5 . FKBP5 knockout cells were generated using CRISPR/Cas9 technology. (A) Representative Western blots in lysates of Jurkat cells with or without the FKBP5 gene treated for 24 hours with DEX (100nM), which robustly induces FKBP5 expression, or vehicle (DMSO). ( B ) Western blots were quantified, and pIKKα levels were normalized to total IKKα levels and are shown as fold changes in comparison to wild-type (FKBP5 +) vehicle-treated cells. The treatment-genotype interaction was tested using two-way ANOVA (F 1,8 = 8.1, interaction p = 2.2 x 10 -2 , n = 3 replicates per condition) and significant effects were followed with -2 Bonferroni-corrected pairwise comparisons. ** p < 10 -2 , statistically significant pairwise comparisons for vehicle vs. DEX. ## p < 10 -2 , statistically significant pairwise comparisons for cells with (wild-type) vs. cells without FKBP5 (knockout).

Article Snippet: The following primary antibodies were used: FLAG (1:7,000, Rockland, 600-401-383), FKBP5/FKBP51 (1:1,000, Bethyl, A301-430A; 1:1000, Cell Signaling, #8245), NF-κB/p65 (1:1000, Cell Signaling, #8242), IKKα (1:1000, Cell Signaling, #2682), pIKKα S176 (1:1000, Cell Signaling, #2078), NIK (1:1000, Cell Signaling, #4994), and Actin (1:5,000, Santa Cruz, sc-1616).

Techniques: Functional Assay, Knock-Out, Generated, CRISPR, Western Blot, Expressing

Journal: bioRxiv

Article Title: Epigenetic derepression of FKBP5 by aging and stress contributes to NF-ĸB-driven inflammation and cardiovascular risk

doi: 10.1101/484709

Figure Lengend Snippet: Annotation of the age- and stress-related FKBP5 CpGs and the constructs used to characterize their function. The figure shows in detail the 5’-3’ sequence upstream of the FKBP5 transcription start site of the DNA stretch (length 224 bp) that was inserted into the CpG-free luciferase reporter vector (ref 63). The black bold letters highlight the sequence of the biotinylated probe (length 70 bp) used for the biotinylated oligonucleotide-mediated chromatin immunoprecipitation. The pink letters highlight the age- and stress-related FKBP5 CpGs. The underlined sequence and label highlight the NF-κB response element (NF-κB RE). As shown, both constructs include the CpGs and response element of interest, while they also completely lack other CpG sites to avoid non-specific CpG methylation effects.

Article Snippet: The following primary antibodies were used: FLAG (1:7,000, Rockland, 600-401-383), FKBP5/FKBP51 (1:1,000, Bethyl, A301-430A; 1:1000, Cell Signaling, #8245), NF-κB/p65 (1:1000, Cell Signaling, #8242), IKKα (1:1000, Cell Signaling, #2682), pIKKα S176 (1:1000, Cell Signaling, #2078), NIK (1:1000, Cell Signaling, #4994), and Actin (1:5,000, Santa Cruz, sc-1616).

Techniques: Construct, Sequencing, Luciferase, Plasmid Preparation, Chromatin Immunoprecipitation, CpG Methylation Assay

Journal: bioRxiv

Article Title: Epigenetic derepression of FKBP5 by aging and stress contributes to NF-ĸB-driven inflammation and cardiovascular risk

doi: 10.1101/484709

Figure Lengend Snippet: (A) Data from dual luciferase reporter gene assays using a CpG-free luciferase reporter vector to which the FKBP5 sequence that surrounds the NF-κB response element was inserted, and which includes the two CpGs of interest but completely lacks other CpG sites (insert sequence shown in Supplementary Fig. 9). This reporter construct was in vitro methylated and transfected into monocyte-derived human cell lines (THP-1). Cells were then stimulated overnight with 25ng/ml Phorbol-12-myristate-13-acetate and 375ng/ml ionomycin (PMA/I), a combination that robustly induces NF-κB signaling. Data are derived from two independent experiments (n = 12 replicates per condition). Comparison was performed using two-way ANOVA with methylation and treatment as factors (F 1,44 = 59.5, interaction p < 10 -3 ), and statistically significant effects were followed with Bonferroni-corrected pairwise comparisons. (B-D) The effect of in vitro DNA methylation on PMA/I-induced NF-κB binding to the NF-κB response element was examined using biotinylated oligonucleotide-mediated chromatin immunoprecipitation (ChIP) in THP-1 cells (oligonucleotide sequence shown in Supplementary Fig. 9). Schematic summary of the experimental setup is shown in B (the lower NF-κB color intensity indicates the expected lower NF-κB binding following in vitro DNA methylation). After ChIP, NF-κB/p65 binding was quantified by Western blotting using antibodies specific for NF-κB (C: example blots; D: quantifications). CTRL (Control) 1: magnetic beads lacking conjugated streptavidin; CTRL 2: cells transfected with non-biotinylated oligonucleotide. Bar graph shows data derived from four independent experiments (t = 2.5, p = 4.4 x 10 -2 , n = 4 per condition). Statistical t-test compared cells carrying the unmethylated probe that were treated overnight with vehicle or PMA/I. Binding was not quantifiable for cells carrying the methylated probe. Data are always shown as fold changes compared to the vehicle-unmethylated cells. Error bars depict the standard error around the group mean. P values for pairwise comparison are shown as follows: *** p < 10 -3 , statistically significant pairwise comparisons for methylated vs. unmethylated. # p < 5 x 10 -2 ; ### p < 10 -3 , statistically significant pairwise comparisons for vehicle vs. drug treatment.

Article Snippet: The following primary antibodies were used: FLAG (1:7,000, Rockland, 600-401-383), FKBP5/FKBP51 (1:1,000, Bethyl, A301-430A; 1:1000, Cell Signaling, #8245), NF-κB/p65 (1:1000, Cell Signaling, #8242), IKKα (1:1000, Cell Signaling, #2682), pIKKα S176 (1:1000, Cell Signaling, #2078), NIK (1:1000, Cell Signaling, #4994), and Actin (1:5,000, Santa Cruz, sc-1616).

Techniques: Luciferase, Plasmid Preparation, Sequencing, Construct, In Vitro, Methylation, Transfection, Derivative Assay, DNA Methylation Assay, Binding Assay, Chromatin Immunoprecipitation, Western Blot, Magnetic Beads

Journal: bioRxiv

Article Title: Epigenetic derepression of FKBP5 by aging and stress contributes to NF-ĸB-driven inflammation and cardiovascular risk

doi: 10.1101/484709

Figure Lengend Snippet: ( A ) Age- and stress-related decrease in FKBP5 DNA methylation is associated with a history of myocardial infarction in two independent cohorts: the KORA, n = 1,648 subjects without vs. 62 with history of MI, β MI = -0.0470, SE = 0.0231, p = 4.1 x 10 -2 , mean DNA methylation difference = 1.8%; and the MPIP, n = 310 subjects without vs. 8 with history of MI, β MI = -0.2300, SE = 0.1177, p = 5.2 x 10 -2 , mean DNA methylation difference = 5.3%; total n = 2,028, meta-analysis p = 1.7 x 10 -2 , heterogeneity p = 1.3 x 10 -1 . The y axis depicts average DNA methylation levels of the two age-regulated FKBP5 CpGs (cg20813374 and cg00130530), after adjusting for confounders (see Methods). Error bars depict the standard error around the group mean. * p < 5 x 10 -2 . ( B ) Schematic summary of study’s findings showing how aging, childhood trauma, and depressive symptoms interact to demethylate FKBP5 at selected promoter CpGs (cg00130530 and cg20813374) located proximally (< 500 bp) upstream the transcription start site (TSS). These epigenetic changes can derepress FKBP5 responses in immune cells, an effect that in turn promotes NF-κB signaling, whereas this is prevented in immune cells concomitantly treated with selective FKBP5 antagonists. Notably, NF-κB signaling is not only activated by FKBP5, but it can also trigger FKBP5 transcription through an NF-κB response element that is flanked and moderated by the age/stress-related CpGs. This forms a positive feedback loop of FKBP5-NF-κB signaling that may be enhanced in individuals with lower methylation at this site. Derepressed FKBP5 responses and NF-κB activity may promote chemotaxis of proinflammatory cells and peripheral inflammation, potentially contributing to cardiovascular risk. KORA, Cooperative Health Research in the Region of Augsburg F4 community study; MPIP, Max Planck Institute of Psychiatry depression case/control study.

Article Snippet: The following primary antibodies were used: FLAG (1:7,000, Rockland, 600-401-383), FKBP5/FKBP51 (1:1,000, Bethyl, A301-430A; 1:1000, Cell Signaling, #8245), NF-κB/p65 (1:1000, Cell Signaling, #8242), IKKα (1:1000, Cell Signaling, #2682), pIKKα S176 (1:1000, Cell Signaling, #2078), NIK (1:1000, Cell Signaling, #4994), and Actin (1:5,000, Santa Cruz, sc-1616).

Techniques: DNA Methylation Assay, Methylation, Activity Assay, Chemotaxis Assay

Journal: Journal of Endocrinology

Article Title: Androgens modulate glucocorticoid receptor activity in adipose tissue and liver

doi: 10.1530/joe-18-0503

Figure Lengend Snippet: Figure 7 AR signaling modulates 11BHSD1 and contributes to GR-regulated transcriptional activity in WAT but not liver. (A, B, C and D) Hsd11b1 mRNA and 11BHSD1 protein expression in WAT and liver upon glucocorticoid and androgen interventions. (E and F) The relationship between CORT levels and the expression of GR-responsive genes Fkbp5, Gilz and Mt2a in WAT and liver. Data are mean ± s.e.m. and N = 6–7 per group. Statistical significance was calculated using a one-way ANOVA with the Tukey multiple-comparisons test. *P < 0.05 vs Vehicle, **P < 0.01 vs Vehicle, $P < 0.05 vs CORT, $$P < 0.01 vs CORT. Representative Western images are shown and quantification of protein expression is based on 6 per group. GAPDH loading controls in Fig. 7D are identical to the ones shown in Fig. 3K.

Article Snippet: The following primary and secondary antibodies were used: 11BHSD1 (in house antibody University of Edinburgh, 1:10), FKBP5 (AB_2103136, R&D systems, AF4094, 1:70), GAPDH (AB_10167668, Santa Cruz Biotechnology, sc25778, 1:50), GR (AB_2631286, Cell Signaling, 12401, 1:10), HPR anti-goat (ProteinSimple, DM006), HRP antirabbit (ProteinSimple, DM001) and HRP anti-sheep (AB_955452, Abcam, ab6900, 1:100).

Techniques: Activity Assay, Expressing, Western Blot

Journal: Nucleic acids research

Article Title: FKBP51 employs both scaffold and isomerase functions to promote NF-κB activation in melanoma.

doi: 10.1093/nar/gkv615

Figure Lengend Snippet: Figure 1. Effect of FKBP51 knockdown on NF-B activation and apoptosis sensitivity in A375 melanoma cells. (A, upper) IB assay of IB phosphory- lation levels in KA samples. IKK complexes were immunoprecipitated from melanoma cells stably knocked down with three different FKBP51 shRNAs (1, 2 and 3) or with a control shRNA (Ctrl) and stimulated with TNF for 15 and 30 min. GST-IB served as substrate (top panel) to measure IKK activity. IB assay also monitored the IKK levels in immunoprecipitated-protein. Whole lysates showed the efficacy of FKBP51 knock down. HSP60 was used as a loading control. The reduced level of GST-p-IB in condition of FKBP51 knock down is consistent with a reduced phosphorylating capacity of immunoprecipitated IKK. (A, lower) EMSA of nuclear extracts of FKBP51-knocked down A375 cells, stimulated with TNF for 30 and 60’. The band was generated by NF-B binding to a 32P-radiolabeled probe. The bands indicated by the arrow are reduced in FKBP51 knock down cells. Nuclear extract was normalized using lamin B as a loading control. The expression of FKBP51 and -actin in total lysates is also shown. (B) Assay of TNF-induced changes in expression levels of IB protein (IB, upper) and mRNA (qPCR, lower). (C and D) Effect of FKBP51 knock down on TNF-induced apop- tosis. (C) Expression levels (protein and mRNA) of pro-apoptotic Bax. (D) Representative flow cytometric histograms of annex-V/PI staining. Data are representative of three independent experiments.

Article Snippet: Antibodies used for co-IP: anti-FKBP51 antibodies were from Novus Biologicals (rabbit polyclonal, NB100-68240) and from Abnova (mouse polyclonal H00002289-B01P, Taipei, Taiwan); anti-IKK antibodies were from Santa Cruz (rabbit polyclonal anti-IKK , H-744; rabbit polyclonal anti-IKK / , H-470).

Techniques: Knockdown, Activation Assay, Immunoprecipitation, Stable Transfection, Control, shRNA, Activity Assay, Generated, Binding Assay, Expressing, Staining

Journal: Nucleic acids research

Article Title: FKBP51 employs both scaffold and isomerase functions to promote NF-κB activation in melanoma.

doi: 10.1093/nar/gkv615

Figure Lengend Snippet: Figure 2. FKBP51 interacts with all IKK complex subunits and supports IKK assembly. (A) Immunoassay of HEK293 cells transfected with several com- binations of expression vectors, namely HA-IKK, -IKK, -IKK, -TAK1 and Flag-FKBP51. Anti-Flag immunoprecipitated proteins were IB assayed with anti-HA and anti-Flag (upper). IB analysis of total lysates is also shown (lower). (B and C) FKBP51 knock down impairs the IKK/IKK/IKK interaction. (B) Immunoassay of HEK293 cells, silenced or not for FKBP51, and transfected with the expression vectors for IKK, HA-IKK and Flag- FKBP51. IKK was immunoprecipitated from cell lysates; IP was analyzed by IB with anti-HA. (C) Endogenous IKK was immunoprecipitated from whole lysates of stably knocked down A375 melanoma cells (1,2) and the relative negetive control (C). Immunoprecipitated proteins were then assayed by IB. Data are representative of three independent experiments.

Article Snippet: Antibodies used for co-IP: anti-FKBP51 antibodies were from Novus Biologicals (rabbit polyclonal, NB100-68240) and from Abnova (mouse polyclonal H00002289-B01P, Taipei, Taiwan); anti-IKK antibodies were from Santa Cruz (rabbit polyclonal anti-IKK , H-744; rabbit polyclonal anti-IKK / , H-470).

Techniques: Transfection, Expressing, Immunoprecipitation, Knockdown, Stable Transfection, Control

Journal: Nucleic acids research

Article Title: FKBP51 employs both scaffold and isomerase functions to promote NF-κB activation in melanoma.

doi: 10.1093/nar/gkv615

Figure Lengend Snippet: Figure 3. FKBP51 isomerase activity affects the enzymatic function of IKK kinase complex. (A) IB of IB phosphorylation levels of samples assayed in KA. IKK complexes were immunoprecipitated from WT and FKBP51-knocked down A375 cells, stimulated with TNF for 30 and 45 min, in the presence or not of FK506. IKK activation was measured using GST-IB as substrate. IB also monitored the expression of IKK in immunoprecipitated protein and FKBP51 in total lysates. HSP60 served as loading control for total lysates. Data are representative of three independent experiments. (B) Immunoassay of HEK293 cells transfected with the HA-IKK and Flag-FKBP51 expression vectors, in the presence or not of FK506. Data are representative of two independent experiments. (C) Study of IB degradation induced by TNF in A375 cells pre-incubated for 1 h in the absence or the presence of FK506, rapamycin and CSA, each compound at the concentration of 0.2 g/ml. Data are representative of two independent experiments. (D) IB degradation induced by TNF- in A375 cells pre-incubated for 1h in the absence or the presence of Rapamycin, CSA and the specific FKBP51 inhibitors SaFit1 and 2, each at the concentration of 0.2 g/ml. Data are representative of two independent experiments. Both specific compounds inhibited TNF--induced IB degradation. (E) Measure by qPCR of CCND1 and FKBP1A levels showed that both SaFit1 and 2 significantly reduced TNF--induced of CCND1 levels of melanoma, in accordance with the NF-B promoting ability of FKBP51. Modulation of cyclin D1 expression level was confirmed by immunoblot. Neither FKBP1A mRNA, nor FKBP12 protein, appeared to be modulated.

Article Snippet: Antibodies used for co-IP: anti-FKBP51 antibodies were from Novus Biologicals (rabbit polyclonal, NB100-68240) and from Abnova (mouse polyclonal H00002289-B01P, Taipei, Taiwan); anti-IKK antibodies were from Santa Cruz (rabbit polyclonal anti-IKK , H-744; rabbit polyclonal anti-IKK / , H-470).

Techniques: Activity Assay, Phospho-proteomics, Immunoprecipitation, Activation Assay, Expressing, Control, Transfection, Incubation, Concentration Assay, Western Blot

Journal: Nucleic acids research

Article Title: FKBP51 employs both scaffold and isomerase functions to promote NF-κB activation in melanoma.

doi: 10.1093/nar/gkv615

Figure Lengend Snippet: Figure 4. FKBP51 regulates interaction between TRAF2 and IKK. (A) Reduced TRAF2 levels in FKBP51 knocked down melanoma cells. QPCR analysis of the TRAF2 and FKBP51 mRNA levels in FKBP51-knocked down A375 cells. Data are representative of three independent experiments. (B) FKBP51 interacts with TRAF2. Immunoassay of HEK293 cells transfected with the HA-TRAF2 and Flag-FKBP51 expression vectors. Data are representative of three independent experiments. (C) FKBP51 silencing prevents TRAF2 interaction with IKK subunits. Immunoassay of whole cell extracts and im- munoprecipitated proteins obtained by HEK293, silenced or not for FKBP51, and transfected with various combinations of HA-IKK, -IKK, -IKK, -TAK1, -TRAF2 and Flag-FKBP51 expression vectors. Data are representative of four independent experiments. (D) FKBP51, IKK subunits and TRAF2 interact each other. Endogenous IKK and FKBP51 were immunoprecipitated from whole lysates of A375 melanoma cells. Immunoprecipitated protein was then assayed in IB. Data are representative of two independent experiments.

Article Snippet: Antibodies used for co-IP: anti-FKBP51 antibodies were from Novus Biologicals (rabbit polyclonal, NB100-68240) and from Abnova (mouse polyclonal H00002289-B01P, Taipei, Taiwan); anti-IKK antibodies were from Santa Cruz (rabbit polyclonal anti-IKK , H-744; rabbit polyclonal anti-IKK / , H-470).

Techniques: Transfection, Expressing, Immunoprecipitation

Journal: Nucleic acids research

Article Title: FKBP51 employs both scaffold and isomerase functions to promote NF-κB activation in melanoma.

doi: 10.1093/nar/gkv615

Figure Lengend Snippet: Figure 5. FKBP51 domains involved in binding to TRAF2 and IKK subunits. (A) Mutation of TPR domain impairs FKBP51/TRAF2 interaction. Immunoassay of HEK293 cells transfected with HA-TRAF2, Flag-FKBP51-mutTPR (carrying point mutation of TPR), FKBP51-mutPPIase (carrying point mutation of PPIase), Myc-Flag-FKBP51s (a truncated FKBP51 isoform lacking of TPR domains). Lysates were subjected to immunoprecipitation with anti-Flag and IB analysis was performed with anti-TRAF2 and anti-Flag (upper). IB of total lysates is also shown (lower). (B) TPR domain of FKBP51 promoted the association between the IKK subunits. Immunoassay of HEK293 cells transfected with the HA-TRAF2, -IKK, -IKK and Flag- FKBP51-mutants expression vectors. Lysates were subjected to immunoprecipitation with anti-Flag, or anti-IKK and analyzed by IB with anti-IKK/ and anti-TRAF2. IB of whole lysates is also shown. (C) Both TPR and PPIase domains of FKBP51 are involved in the IKK/FKBP51 interaction. Immunoassay of HEK293 cells transfected with HA-IKK and differents Flag-FKBP51 mutants. Lysates were subjected to immunoprecipitation with anti-IKK and analyzed by IB with anti-Flag. IB analysis of whole lysates is also shown. Data are representative of three independent experiments.

Article Snippet: Antibodies used for co-IP: anti-FKBP51 antibodies were from Novus Biologicals (rabbit polyclonal, NB100-68240) and from Abnova (mouse polyclonal H00002289-B01P, Taipei, Taiwan); anti-IKK antibodies were from Santa Cruz (rabbit polyclonal anti-IKK , H-744; rabbit polyclonal anti-IKK / , H-470).

Techniques: Binding Assay, Mutagenesis, Transfection, Immunoprecipitation, Expressing

Journal: Nucleic acids research

Article Title: FKBP51 employs both scaffold and isomerase functions to promote NF-κB activation in melanoma.

doi: 10.1093/nar/gkv615

Figure Lengend Snippet: Figure 6. Proposed mechanism for the interaction of FKBP51 with NF-B signaling proteins. (A) TNF-binding to its receptor determines formation of RIP-induced K-63 ubiquitin chain. (B) TRAF2, promotes elongation of this non degradative ubiquitin chain and recruits TAK1 kinase complex. (C) TRAF2 interacts with TPR domain of FKBP51. IKK interacts with both FK and TPR domains of FKBP51. IKK and TRAF2 are also connected through K63 ubiquitin chain. (D) IKK and interact each others and with IKK and TRAF2 through the TPR domain.

Article Snippet: Antibodies used for co-IP: anti-FKBP51 antibodies were from Novus Biologicals (rabbit polyclonal, NB100-68240) and from Abnova (mouse polyclonal H00002289-B01P, Taipei, Taiwan); anti-IKK antibodies were from Santa Cruz (rabbit polyclonal anti-IKK , H-744; rabbit polyclonal anti-IKK / , H-470).

Techniques: Binding Assay, Ubiquitin Proteomics

Journal: eLife

Article Title: Overcoming mutation-based resistance to antiandrogens with rational drug design

doi: 10.7554/eLife.00499

Figure Lengend Snippet: LNCaP-Pb.PSE.EGFP cells overexpressing AR WT and cells from the fifth sort of our screen were transfected with 10 nM of either a non-targeting siRNA (siNT) or a siRNA against AR (siAR). They were also treated with either vehicle (V) or 1 μM enzalutamide (E). After 4 days of enzalutamide treatment and siRNA knockdown, cells were collected for both ( A ) flow cytometric analysis of EGFP expression and ( B ) western blot analysis of the AR target gene FKBP5, and to ensure we achieved good AR knockdown. AR: androgen receptor; WT: wild-type. DOI: http://dx.doi.org/10.7554/eLife.00499.006

Article Snippet: Antibodies used for immunoblot assays were β-actin (AC-15; Sigma) PARP (#9541; Cell Signaling Technology, Danvers, MA), FKBP5 (IHC-00289; Bethyl, Montgomery, TX), β-tubulin (D-10), and androgen receptor (N-20) (both from Santa Cruz Biotechnology, Dallas, TX).

Techniques: Transfection, Knockdown, Expressing, Western Blot

Journal: eLife

Article Title: Overcoming mutation-based resistance to antiandrogens with rational drug design

doi: 10.7554/eLife.00499

Figure Lengend Snippet: ( A ) A representative FACS histogram shows the induction of AR-dependent EGFP expression by enzalutamide in LNCaP-Pb.PSE.EGFP cells ectopically expressing AR F876L. The magnitude of induction by enzalutamide (10 μM) is comparable to that conferred by the endogenous androgen DHT (1 nM). Enzalutamide treatment of LNCaP-Pb.PSE.EGFP cells ectopically expressing AR WT effectively suppressed EGFP expression. Geometric-mean fluorescence intensity for WT treated cells: vehicle (348), enzalutamide (66.4), DHT (1554); for F876L cells: vehicle (345), enzalutamide (1051), DHT (1699). ( B ) Cotransduction of CV1 cells with an AR-regulated firefly luciferase construct, a constitutive Renilla luciferase construct, and one of the indicated AR constructs, recapitulates the pharmacology observed in the EGFP reporter system. These cells were treated with vehicle (DMSO), antiandrogens (1 μM), or the synthetic androgen R1881 (1 nM). A dual luciferase assay was conducted on cell lysates, the firefly signal was normalized to the constitutive Renilla activity, and the data are reported as relative light units (RLUs). Notably, the bisaryl-thiohydantoin antiandrogens (enzalutamide and ARN-509) effectively induce AR F876L transcriptional activity, while structurally discrete antiandrogens (hydroxyflutamide and bicalutamide) do not impact AR F876L activity in this assay. As expected, the transcriptional activity of AR W741C or AR T877A was induced by bicalutamide or hydroxyflutamide, respectively. ( C ) Quantitative reverse transcription–polymerase chain reaction analysis of LNCaP/AR F876L cells shows that enzalutamide (1 μM) can induce the expression of canonical AR-regulated gene products (i.e., PSA , TMPRSS2 , SGK1 , and FKBP5 ). Relative gene expression post therapy for LNCaP/AR WT cells is included as positive controls. FL = AR F876L, data are normalized to GAPDH and represented as mean ± SD, n = 3. ( D ) Cell proliferation data shows that overexpression of AR F876L in a human prostate cancer cell line sensitive to enzalutamide therapy can rescue cell growth. VCaP cells overexpressing either AR WT (solid lines) or AR F876L (dashed lines) were cultured in media containing full serum, treated with either vehicle (DMSO) or 10 μM enzalutamide, and the viable cell fraction was determined at the indicated time points (data is represented as mean ± SD, n = 3). ( E ) Cellular proliferation data shows that enzalutamide also rescues the growth of VCaP cells expressing AR F876L in androgen-depleted media. VCaP cells overexpressing either AR WT (solid lines) or AR F876L (dashed lines) were treated with vehicle (DMSO), 1 nM DHT, or 10 μM enzalutamide, and the viable cell fraction was determined at the indicated time points (mean ± SD, n = 3). ( F ) A time to progression study for mice bearing subcutaneous LNCaP/AR-WT (solid lines) or LNCaP/AR-F876L (dashed lines) xenografts further highlights the genotype-dependent pharmacology of enzalutamide. Inoculated animals were treated once daily through oral gavage with either vehicle or enzalutamide (30 mg/kg), and tumor size was monitored weekly (11–16 tumors per treatment group). While enzalutamide potently suppressed the growth of LNCaP/AR-WT tumors, LNCaP/AR-F876L tumors exposed to enzalutamide grew with kinetics roughly equivalent to either vehicle treatment arm. AR: androgen receptor; WT: wild-type. DOI: http://dx.doi.org/10.7554/eLife.00499.008

Article Snippet: Antibodies used for immunoblot assays were β-actin (AC-15; Sigma) PARP (#9541; Cell Signaling Technology, Danvers, MA), FKBP5 (IHC-00289; Bethyl, Montgomery, TX), β-tubulin (D-10), and androgen receptor (N-20) (both from Santa Cruz Biotechnology, Dallas, TX).

Techniques: Expressing, Fluorescence, Luciferase, Construct, Activity Assay, Reverse Transcription, Polymerase Chain Reaction, Gene Expression, Over Expression, Cell Culture

Journal: eLife

Article Title: Overcoming mutation-based resistance to antiandrogens with rational drug design

doi: 10.7554/eLife.00499

Figure Lengend Snippet: ( A ) LNCaP cells were transfected with EYFP-tagged wild-type AR or AR F876L in androgen depleted media containing vehicle, 1 μM enzalutamide, or 1 nM DHT. Representative confocal images are shown. Average nuclear-to-cytoplasmic ratios for EYFP are displayed (±SD, n = 3). ( B ) LNCaP cells stably overexpressing either AR WT or AR F876L were cultured in androgen-depleted media for 4 days, then treated with vehicle (VEH), 10 μM enzalutamide (ENZ), or 1 nM DHT for 4 hr. AR chromatin immuoprecipitation was performed, and real-time PCR quantification of PSA enhancer and FKBP5 enhancer is shown (percent input mean ± SD, n = 3). AR: androgen receptor; WT: wild-type. DOI: http://dx.doi.org/10.7554/eLife.00499.010

Article Snippet: Antibodies used for immunoblot assays were β-actin (AC-15; Sigma) PARP (#9541; Cell Signaling Technology, Danvers, MA), FKBP5 (IHC-00289; Bethyl, Montgomery, TX), β-tubulin (D-10), and androgen receptor (N-20) (both from Santa Cruz Biotechnology, Dallas, TX).

Techniques: Transfection, Stable Transfection, Cell Culture, Real-time Polymerase Chain Reaction