Journal: eLife

Article Title: Computational and cellular studies reveal structural destabilization and degradation of MLH1 variants in Lynch syndrome

doi: 10.7554/eLife.49138

Figure Lengend Snippet:

Article Snippet: Recombinant DNA reagent , pCMV6-MYC-DDK-HSP70-1A (HSPA1A) , OriGene , RC200270 , -.

Techniques: Recombinant, Software

Journal: Pharmacological research

Article Title: Allosteric inhibition of HSP70 in collaboration with STUB1 augments enzalutamide efficacy in antiandrogen resistant prostate tumor and patient-derived models

doi: 10.1016/j.phrs.2023.106692

Figure Lengend Snippet: JG98 degrades AR-V7 and suppresses HSP70 induced AR-V7 transcriptional activity A. Whole cell lysates of CWR22Rv1 and C4–2B MDVR cells after 24 h treatment with JG98 (0, 2.5 or 5 μM) were separated by electrophoresis and probed for AR-V7, AR-FL, and HSP70 with their respective antibodies. GAPDH was used as the internal control. B. CWR22Rv1 and C4–2B MDVR cells were treated with DMSO, JG98 (0.25 μM), enzalutamide (20 μM), or the combination for 5 days and the levels of AR-V7 and AR-FL were examined by western blotting. GAPDH was used as the internal control. C. HEK293 cells transfected with the AR-V7 expression construct were treated with or without JG98 (2.5 μM) and immunoprecipitated with anti-AR antibodies and probed for ubiquitin, AR-V7, and HSP70, respectively. IgG antibodies were used as the negative control and whole lysate input were loaded alongside. D. CWR22Rv1 cells with the endogenous AR/AR-V7 were treated with or without JG98 (2.5 μM) for 24 h. Cell lysates were immunoprecipitated with anti-AR antibodies and probed for ubiquitin, AR-V7, AR-FL, and HSP70, respectively. E. CWR22Rv1 cells were treated with 50 μg/ml cycloheximide in the absence or presence of 5 μM of JG98. Total cell lysates were collected 0, 2, 4, 8, and 24 h after treatment. AR-V7 and AR-FL were analyzed by western blotting to calculate the half-life of AR-V7 and AR-FL. F. CWR22Rv1 and C4–2B MDVR cells were treated with or without JG98 (2.5 μM) in the absence or presence of the proteosome inhibitor, MG132 (5 μM), for additional 8 h, and the protein expression of AR-V7 and AR-FL were analyzed by western blotting. G. HEK293 cells were transiently transfected with pcDNA, HSP70, AR-V7, or the combination constructs with PSA-Luc, and treated with control or 1 nM DHT. Cell lysates were harvested 24 h after treatments, and the PSA luciferase activity was assessed. H. HEK293 cells were transiently transfected with vector only, AR-V7, or AR-V7+HSP70 expressing plasmids with PSA-Luc, and subsequently treated with DMSO, JG98 (2.5, 5 μM) or enzalutamide (20 μM). PSA luciferase activity was measured. * p < 0.05. Results are the mean of three independent experiments (± S.D.). ns: not significant.

Article Snippet: Cells were transiently transfected with plasmids expressing AR-FL [ ], AR-V7 [ ], Flag-STUB1 (Sino Biological, Catalog# HG12496-NF), or HSP70 (HSPA1B, OriGene, Catalog# SC116767), using Lipofectamine 2000 (Invitrogen).

Techniques: Activity Assay, Electrophoresis, Control, Western Blot, Transfection, Expressing, Construct, Immunoprecipitation, Ubiquitin Proteomics, Negative Control, Luciferase, Plasmid Preparation

Journal: Pharmacological research

Article Title: Allosteric inhibition of HSP70 in collaboration with STUB1 augments enzalutamide efficacy in antiandrogen resistant prostate tumor and patient-derived models

doi: 10.1016/j.phrs.2023.106692

Figure Lengend Snippet: JG98 promotes AR-V7 degradation through STUB1 A. HEK293 cells were co-transfected with AR-V7, HSP70, and Flag-STUB1 for 3 days and then treated with 2.5 μM JG98 for 24 h. The cells were then permeabilized with paraformaldehyde and probed with anti-AR-V7 and anti-Flag antibodies, respectively. Location of AR-V7 was visualized with FITC-conjugated and Flag-STUB1 with Alexa467-conjugated secondary antibodies. Nuclei were stained with DAPI. Merged images displayed interaction between AR-V7 and Flag-STUB1 under JG98 treatment. White arrows indicate the typical staining of cells in each group. Scale bar 20 μm. B. C4–2B MDVR cells treated with STUB1 siRNA or control and treated with various doses of JG98 (0, 1, 2.5 μM) for 24 h. Whole cell lysates were separated by electrophoresis and blotted with AR-V7, AR-FL and STUB1 antibodies. Levels of tubulin were assessed for loading equity. C. C4–2B MDVR cells were transfected with siRNA against STUB1 or control for 5 days. Cell viability was determined by cell counting and represented as cell survival rates. * p < 0.05. Results are the mean of three independent experiments (± S.D.).

Article Snippet: Cells were transiently transfected with plasmids expressing AR-FL [ ], AR-V7 [ ], Flag-STUB1 (Sino Biological, Catalog# HG12496-NF), or HSP70 (HSPA1B, OriGene, Catalog# SC116767), using Lipofectamine 2000 (Invitrogen).

Techniques: Transfection, Staining, Control, Electrophoresis, Cell Counting