Journal: Scientific Reports

Article Title: Splenic glucocorticoid resistance following psychosocial stress requires physical injury

doi: 10.1038/s41598-017-15897-2

Figure Lengend Snippet: Effects of 19 days of chronic subordinate colony housing (CSC) on the spleen, in the presence and absence of CD11b + -cells. Depicted is the absolute spleen weight [mg] ( A ) and the number of splenocytes per spleen ( B ) following 20 days of CSC (n = 48) and single-housing (single housed controls, SHC; n = 48). Splenocytes were cultured without (basal) and with lipopolysaccharide (LPS, 1 µg/ml) in presence of various corticosterone (CORT) concentrations. After 48 h of incubation, cell viability was measured. Depicted is the cell viability [optical density (OD)] under basal and LPS-stimulated conditions in the absence of CORT ( C ) and the basal cell viability (without LPS) in the presence of different CORT concentrations (CORT = 0 µM was set to 100%; D ). Delta cell viability of LPS-stimulated minus respective basal wells at CORT = 0 µM is depicted in ( E ). Shown is further the delta cell viability [OD] (LPS-stimulated minus respective basal wells) at various CORT concentrations in percent of respective values at CORT = 0 µM (set to 100%; F ). Depicted are further the absolute numbers of CD19 + , CD3 + CD4 + , CD3 + CD8 + and CD11b + cells within total viable splenocytes ( G ) of another set of SHC (n = 8) and CSC (n = 8) mice. CD11b + as well as CD11b − cells from these mice were cultured without (basal) and with LPS (1 µg/µl) in absence or presence of various CORT concentrations. Depicted is the cell viability under basal and LPS-stimulated conditions ( H ) as well as delta cell viability of LPS-stimulated minus respective basal wells ( I ) of CD11b − and CD11b + splenocytes. Shown is further the delta cell viability (LPS-stimulated minus respective basal wells) of CD11b − splenocytes at various CORT concentrations in percent of respective values at CORT = 0 µM (set to 100%; J ). A third set of SHC (n = 11) and CSC (n = 10) mice was employed to determine relative protein expression of glucocorticoid receptor (GR; K ) and FK506 binding protein 51 (FKBP51; L ) [grey density] on day 20 of CSC using western blotting, all normalized to the loading control glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Representative images of bands detected for GR (~86 kDa) and FKBP51 (~51 kDa) and respective loading control GAPDH (~36 kDa) are shown for SHC and CSC mice below each subfigure. SHC; CSC. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 versus respective SHC; # P ≤ 0.05, ## P ≤ 0.01, ### P ≤ 0.001 versus respective basal (CORT = 0 µM) values.

Article Snippet: Western blotting was performed as described previously , , using equal amounts of protein lysates (20 μg) and antibody for rabbit anti-mouse GR (1:1500, Santa Cruz Biotechnology, Inc., Heidelberg, Germany) and goat anti-mouse FKBP51 (1:500, Santa Cruz Biotechnology, Inc., Heidelberg, Germany).

Techniques: Cell Culture, Incubation, Expressing, Binding Assay, Western Blot, Control

Journal: Cell Death Discovery

Article Title: The inhibition of FKBP5 protects β-cell survival under inflammation stress via AKT/FOXO1 signaling

doi: 10.1038/s41420-023-01506-x

Figure Lengend Snippet: A mRNA expression of FKBP5 in human islets from 5 organ donors treated with proinflammatory cytokines (IL-1β 10 ng/mL, TNF-α 25 ng/mL, IFN-γ 100 ng/mL). Experiments were repeated for 3 times. B , C mRNA expression of Fkbp5 in NIT-1 cells ( B ) and βTC-6 cells ( C ). Experiments were repeated for 3 times. D , E Western blot of Fkbp5 in NIT-1 cells treated with proinflammatory cytokines ( D ) and Fkbp5 expression levels were quantified by Image J, with β-actin as loading control. E Experiments were repeated for 3 times. Student’s t -test. Mean ± SEM, * P ＜0.05, ** P ＜0.01, *** P <0.001.

Article Snippet: Immunohistochemical staining was performed using FKBP5 (1:300, ABclonal, OH, USA) and p-Foxo1 (1:300, Novus Biological, Cambridge, UK), then incubation with TRITC AffiniPure Goat Anti-Rabbit IgG H&L (1:200, Jackson Immunoresearch Laboratories and Molecular Probes, West Grove, PA, USA) secondary antibodies.

Techniques: Expressing, Western Blot, Control

Journal: Cell Death Discovery

Article Title: The inhibition of FKBP5 protects β-cell survival under inflammation stress via AKT/FOXO1 signaling

doi: 10.1038/s41420-023-01506-x

Figure Lengend Snippet: A , B Western blot analysis of FKBP5, p-FOXO1 Ser256 , FOXO1, p-AKT Ser473 , and AKT in primary human islets transfected with siFKBP5 or control siRNAs for 48 h ( A ). Relative proteins expression levels were quantified by Image J ( B ). C , D Western blot analysis of p-FOXO1 Ser256 , FOXO1, p-AKT Ser473 , and AKT expressions in primary human islets treated with SAFit2 or control vehicle for 48 h ( C ). Relative proteins expression levels were quantified by Image J ( D ). E , F Western blot analysis of p-Foxo1 Ser256 , Foxo1, p-Akt Ser473 , Akt and Fkbp5 in NIT-1 cells treated with SAFit2 (1 µM) for 0, 0.5, 1, 2, 4, 8 h ( E ). Relative proteins expression levels were quantified by Image J ( F ) . G , H Immunofluorescence staining with p-Foxo1 (red) ( G ) or Fkbp5 (red) ( H ), insulin (Ins, green) and DAPI (blue) in NIT-1 cells treated with SAFit2 or control vehicle for 24 h. Scale: 20 μm. I – K Western blot analysis nuclear ( I ) and cytoplasmic ( J ) protein of p-Foxo1 Ser256 and Foxo1 in NIT-1 cells treated with SAFit2 or control vehicle. Relative proteins expression levels were quantified by Image J ( K ). L , M Western blot analysis of p-Foxo1 Ser256 , Foxo1, p-Akt Ser473 , Akt, expression in NIT-1 cells treated with proinflammatory cytokines, or Cytokines + SAFit2 ( L ). Relative proteins expression levels were quantified by Image J ( M ). Experiments were repeated for 3 times. Student’s t -test. Mean ± SEM, * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Immunohistochemical staining was performed using FKBP5 (1:300, ABclonal, OH, USA) and p-Foxo1 (1:300, Novus Biological, Cambridge, UK), then incubation with TRITC AffiniPure Goat Anti-Rabbit IgG H&L (1:200, Jackson Immunoresearch Laboratories and Molecular Probes, West Grove, PA, USA) secondary antibodies.

Techniques: Western Blot, Transfection, Control, Expressing, Immunofluorescence, Staining

Journal: Cell Death Discovery

Article Title: The inhibition of FKBP5 protects β-cell survival under inflammation stress via AKT/FOXO1 signaling

doi: 10.1038/s41420-023-01506-x

Figure Lengend Snippet: A Immunofluorescence staining with FKBP5 and insulin in human pancreatic tissues of ND and T2D subjects. n = 3. Red: FKBP5, Green: Insulin,.Blue: DAPI. Scale: 50 μm. B Immunofluorescence staining with Fkbp5 and insulin in mice pancreatic tissues of C57BL/6J and db/db mice. n = 3. Red: Fkbp5, Green: Insulin, Blue: DAPI. Scale: 50 μm. C Immunofluorescence staining with pFoxo1 and insulin in mice pancreatic tissues of C57BL/6J and db/db mice. n = 3. Red: pFoxo1, Green: Insulin, Blue: DAPI. Scale: 50 μm.

Article Snippet: Immunohistochemical staining was performed using FKBP5 (1:300, ABclonal, OH, USA) and p-Foxo1 (1:300, Novus Biological, Cambridge, UK), then incubation with TRITC AffiniPure Goat Anti-Rabbit IgG H&L (1:200, Jackson Immunoresearch Laboratories and Molecular Probes, West Grove, PA, USA) secondary antibodies.

Techniques: Immunofluorescence, Staining

Journal: PLoS ONE

Article Title: Org 214007-0: A Novel Non-Steroidal Selective Glucocorticoid Receptor Modulator with Full Anti-Inflammatory Properties and Improved Therapeutic Index

doi: 10.1371/journal.pone.0048385

Figure Lengend Snippet: A) In a co-factor recruitment assay, Org 214007-0, in comparison to prednisolone, shows potent but partial recruitment of a 0.1 μM peptide presenting TIF2 -3. On the Y-axis average fluorescence counts (+/− SD) are shown. EC50 values (%CV) and percentages maximal efficacy (%CV) for Org 214007-0 versus prednisolone were 10 (7.4) nM versus 48 (3.2) nM and 67 (7.1) % vs 100% respectively. B) In THP1 cells Org 214007-0 shows partial induction of FKBP51 protein expression and C) under inflammatory conditions represses the IL-6 protein expression almost as good as prednisolone does.

Article Snippet: For the FKBP51-specific AlphaLISA anti-FKBP51 Mab (Abnova, Cat H0002289-M01) and Goat anti-IgG FKBP51 polyclonal (Santa Cruz Biotechnology, Cat sc-11514) were used and the same procedure was followed as for the cytokine-specific AlphaLISAs (more detailed information in Supportive Information) (SI text).

Techniques: Comparison, Fluorescence, Expressing

Journal: PLoS ONE

Article Title: Org 214007-0: A Novel Non-Steroidal Selective Glucocorticoid Receptor Modulator with Full Anti-Inflammatory Properties and Improved Therapeutic Index

doi: 10.1371/journal.pone.0048385

Figure Lengend Snippet: Fold changes for the top 25 genes either induced (A) or repressed (B) by 1 μM prednisolone and 1 μM Org 214007-0 in THP-1 cells. NB. Scales are in 2log. Example of an induced gene, FK506 binding protein 51 (FKBP51) (C) and a repressed gene, interleukin 6 (IL-6) (D) in comparison to the vehicle control under either non-stimulated or stimulated (IFNγ/TNFα) condition. Fold changes for the top 25 genes either induced (E) or repressed (F) by 1.5 mg/kg prednisolone and 0.3 mg/kg Org 214007-0 in muscle tissue from arthritic mice. NB. Scales are in 2log. Example of an induced gene (Per-2) (G) and a repressed gene (Ccl8) (H) in comparison to vehicle treated arthritic mice and vehicle treated healthy mice.

Article Snippet: For the FKBP51-specific AlphaLISA anti-FKBP51 Mab (Abnova, Cat H0002289-M01) and Goat anti-IgG FKBP51 polyclonal (Santa Cruz Biotechnology, Cat sc-11514) were used and the same procedure was followed as for the cytokine-specific AlphaLISAs (more detailed information in Supportive Information) (SI text).

Techniques: Binding Assay, Comparison, Control

Journal: PLoS ONE

Article Title: Org 214007-0: A Novel Non-Steroidal Selective Glucocorticoid Receptor Modulator with Full Anti-Inflammatory Properties and Improved Therapeutic Index

doi: 10.1371/journal.pone.0048385

Figure Lengend Snippet: Summary of studies to define the therapeutic index (TI) of Org 214007-0.

Article Snippet: For the FKBP51-specific AlphaLISA anti-FKBP51 Mab (Abnova, Cat H0002289-M01) and Goat anti-IgG FKBP51 polyclonal (Santa Cruz Biotechnology, Cat sc-11514) were used and the same procedure was followed as for the cytokine-specific AlphaLISAs (more detailed information in Supportive Information) (SI text).

Techniques:

Journal: PLoS ONE

Article Title: Org 214007-0: A Novel Non-Steroidal Selective Glucocorticoid Receptor Modulator with Full Anti-Inflammatory Properties and Improved Therapeutic Index

doi: 10.1371/journal.pone.0048385

Figure Lengend Snippet: A) Ratio of the read count for clusters induced by 1 μM prednisolone or 1 μM Org 214007-0 in a ChIP-Seq analysis. If Org 214007-0 and prednisolone would induce an equal number of reads, the histogram would be centered on Log0, indicated by the dotted line. There is a clear shift to the right from this line (P-value (mean = 0) <0.000001), indicating that prednisolone leads to more GR occupancy than Org 214007-0. B) Example profile of the tag clusters in the GR response gene FKBP51. Multiple binding sites are found within this gene, each showing denser clusters after treatment with 1 μM prednisolone than after treatment with 1 μM Org 214007-0. The inset highlights an intronic region at 87 kb downstream of the transcription start site. This region that was identified by Paakinaho et al. (2010) , as a major intronic enhancer in human A459 lung cancer cells and was shown to be occupied by GR after treatment with the GR ligand dexamethasone.

Article Snippet: For the FKBP51-specific AlphaLISA anti-FKBP51 Mab (Abnova, Cat H0002289-M01) and Goat anti-IgG FKBP51 polyclonal (Santa Cruz Biotechnology, Cat sc-11514) were used and the same procedure was followed as for the cytokine-specific AlphaLISAs (more detailed information in Supportive Information) (SI text).

Techniques: ChIP-sequencing, Binding Assay