thiolutin  (MedChemExpress)

Journal: Theranostics

Article Title: Coupling proteostasis and de novo purine biosynthesis of PSMD14 fuels glioblastoma progression and chemoresistance

doi: 10.7150/thno.124409

Figure Lengend Snippet: PSMD14 regulates IMPDH2 stability through selective removal of K48-linked ubiquitin chains. (A-C) Western blotting analysis of ubiquitin conjugates in LN229 cells with PSMD14 knockdown. (D-F) Western blotting analysis of ubiquitin conjugates in GBM#P3 cells with PSMD14 knockdown. (G, H) Ubiquitination status of IMPDH2 upon PSMD14 inhibition. GBM cells were treated with the PSMD14 inhibitor thiolutin in the presence of MG132. (I-L) Domain mapping of PSMD14's deubiquitinating function. GBM cells were co-transfected with wild-type PSMD14 or N-terminal truncation mutants along with plasmids encoding Myc-tagged ubiquitin.

Article Snippet: Where specified, cells were pre-treated with cycloheximide (25 μg/mL for 9 h; HY-123320; MedChemExpress), MG132 (10 μM for 8 h; #474790; Sigma-Aldrich), or thiolutin (2 μM for 8 h; HY-N6712; MedChemExpress) prior to lysis.

Techniques: Ubiquitin Proteomics, Western Blot, Knockdown, Inhibition, Transfection

Journal: Theranostics

Article Title: Coupling proteostasis and de novo purine biosynthesis of PSMD14 fuels glioblastoma progression and chemoresistance

doi: 10.7150/thno.124409

Figure Lengend Snippet: Pharmacological inhibition of PSMD14 with thiolutin reduces tumor burden and synergizes with temozolomide in GBM. (A) IC50 curves of Thiolutin in the GBM#P3, U118, and LN229 cell lines. (B) Time-course of cell viability in GBM cells with ectopic PSMD14 overexpression or empty vector control, in the presence of DMSO or thiolutin. (C) Quantification of cell viability (OD values) on Day 4 (n = 3). ***P <0.001 (one-way ANOVA). (D) Representative bioluminescence images of intracranial GBM#P3 xenograft-bearing mice at Days 7 and 28 post-implantation. Mice were treated with vehicle or thiolutin, with or without PSMD14 overexpression in the implanted cells. (E) Quantification of total photon flux from intracranial tumors at Day 28. Data are presented as mean ± SD (n = 5 per group). ***P <0.001 (one-way ANOVA). (F) Kaplan-Meier survival analysis of mice bearing intracranial tumors under the indicated treatments (n = 10 per group). (G) Representative HE is staining of brain sections from tumor-bearing mice across treatment groups. Scale bar = 2.5 mm. (H) IHC staining for PSMD14 and IMPDH2 in brain tumor sections from the orthotopic xenografts. Scale bar = 100 μm. (I) Representative IF images of tumor tissues from the xenograft models. Scale bar = 100 μm. (J) Cell viability curves for GBM cells treated for 4 days with vehicle (DMSO), TMZ, thiolutin, or the combination of TMZ + thiolutin. (K) Quantification of cell viability (OD) at Day 4. Data are presented as mean ± SD (n = 3). ***P <0.001 (one-way ANOVA). (L) Bioluminescent imaging of intracranial tumors in mice treated with vehicle, TMZ, or TMZ + thiolutin on Days 7 and 28 post-implantation. (M) Quantification of photon flux at Day 28. Data are presented as mean ± SD (n = 5 per group). *P < 0.05, **P < 0.01, ***P < 0.001 (one-way ANOVA). (N) Representative IHC images of PSMD14 and IMPDH2 in tumor tissues from mice treated with TMZ alone or TMZ + thiolutin. Scale bar = 100 μm.

Article Snippet: Where specified, cells were pre-treated with cycloheximide (25 μg/mL for 9 h; HY-123320; MedChemExpress), MG132 (10 μM for 8 h; #474790; Sigma-Aldrich), or thiolutin (2 μM for 8 h; HY-N6712; MedChemExpress) prior to lysis.

Techniques: Inhibition, Over Expression, Plasmid Preparation, Control, Staining, Immunohistochemistry, Imaging