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: 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