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 interacts with IMPDH2 and regulates its protein stability in GBM. (A) Western blot confirming overexpression of exogenous Flag-tagged PSMD14 in U118MG cells. (B) The Co-IP assay of PSMD14 from U118MG cells. (C) Mass spectrometry identification of proteins co-precipitated with PSMD14 in U118MG cells, followed by intersection analysis to identify common candidate interactors. (D) Bioinformatic overlap of the PSMD14 interactome identified by mass spectrometry with known interactions in the BioGRID database. (E) Network analysis of high-confidence interacting proteins with PSMD14. (F) Western blotting analysis to detect changes in IMPDH2 expression after knockdown of PSMD14. (G) The Co-IP assay confirming the interaction between PSMD14 and IMPDH2 in GBM cells. (H) Structural docking model of PSMD14 and IMPDH2 showing the predicted binding interface rendered with PyMOL (PSMD14 in pink, IMPDH2 in purple). (I) Schematic diagram of the PSMD14 and IMPDH2 domain structures and the design of truncation mutants. (J) The Co-IP analysis using PSMD14 and IMPDH2 truncation mutants, identifying the regions required for their interaction. (K) Cycloheximide (CHX) chase assay in control and PSMD14-knockdown GBM cells, evaluating IMPDH2 protein stability over time. (L) Quantification of IMPDH2 protein half-life from CHX chase assays. Data are presented as mean ± SD (n = 3). *P < 0.05 (one-way ANOVA).

Article Snippet: Membranes were blocked in 5% (w/v) non-fat milk in TBST for 1 h, then incubated overnight at 4 °C with primary antibodies targeting PSMD14 (4197S; 1:1,000; Cell Signaling Technology), IMPDH2 (36281S; 1:1,000; Cell Signaling Technology), β-Actin (4970S; 1:1,000; Cell Signaling Technology), ubiquitin (20326S; 1:1,000; Cell Signaling Technology), K48-linkage-specific polyubiquitin (8081S; 1:1,000; Cell Signaling Technology), K63-linkage-specific polyubiquitin (5621S; 1:1,000; Cell Signaling Technology), His-Tag (12698T; 1:1000; Cell Signaling Technology), HA-Tag (3724S; 1:1000; Cell Signaling Technology) and Myc-Tag (2276S; 1:1,000; Cell Signaling Technology).

Techniques: Western Blot, Over Expression, Co-Immunoprecipitation Assay, Mass Spectrometry, Expressing, Knockdown, Binding Assay, Control

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: Membranes were blocked in 5% (w/v) non-fat milk in TBST for 1 h, then incubated overnight at 4 °C with primary antibodies targeting PSMD14 (4197S; 1:1,000; Cell Signaling Technology), IMPDH2 (36281S; 1:1,000; Cell Signaling Technology), β-Actin (4970S; 1:1,000; Cell Signaling Technology), ubiquitin (20326S; 1:1,000; Cell Signaling Technology), K48-linkage-specific polyubiquitin (8081S; 1:1,000; Cell Signaling Technology), K63-linkage-specific polyubiquitin (5621S; 1:1,000; Cell Signaling Technology), His-Tag (12698T; 1:1000; Cell Signaling Technology), HA-Tag (3724S; 1:1000; Cell Signaling Technology) and Myc-Tag (2276S; 1:1,000; Cell Signaling Technology).

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: Loss of PSMD14 disrupts nucleotide metabolism, induces DNA damage and impairs mitochondrial function in GBM cells. (A) Schematic depiction of metabolic pathways regulated by IMPDH2 in GBM cells. (B-D) Relative levels of metabolites in GBM cells expressing siIMPDH2 or shPSMD14. Data are presented as mean ± SD (n = 3). **P < 0.01, ***P < 0.001 (one-way ANOVA). (E) ICC images showing accumulation of γ-H2AX in GBM cells upon PSMD14 knockdown. Scale bar = 30 μm. (F) ICC images indicating nucleolar stress in PSMD14-knockdown GBM cells. Nucleoli are labeled by nucleostemin. Scale bar = 30 μm. (G) ICC images of mitochondria in PSMD14-knockdown cells. Scale bar = 30 μm. (H) Seahorse extracellular flux analysis of mitochondrial respiration in control and PSMD14-knockdown GBM cells. Data are presented as mean ± SD (n = 3). *P < 0.05, **P < 0.01 (Independent-sample Student-T test). (I) Flow cytometric analysis of mitochondrial membrane potential in control and PSMD14-knockdown cells. Data are presented as mean ± SD (n = 3). *P < 0.05, **P < 0.01 (Independent-sample Student-T test). (J-K) ICC images of JC-1 staining showing changes in mitochondrial membrane potential. Scale bar = 30 μm.

Article Snippet: Membranes were blocked in 5% (w/v) non-fat milk in TBST for 1 h, then incubated overnight at 4 °C with primary antibodies targeting PSMD14 (4197S; 1:1,000; Cell Signaling Technology), IMPDH2 (36281S; 1:1,000; Cell Signaling Technology), β-Actin (4970S; 1:1,000; Cell Signaling Technology), ubiquitin (20326S; 1:1,000; Cell Signaling Technology), K48-linkage-specific polyubiquitin (8081S; 1:1,000; Cell Signaling Technology), K63-linkage-specific polyubiquitin (5621S; 1:1,000; Cell Signaling Technology), His-Tag (12698T; 1:1000; Cell Signaling Technology), HA-Tag (3724S; 1:1000; Cell Signaling Technology) and Myc-Tag (2276S; 1:1,000; Cell Signaling Technology).

Techniques: Expressing, Knockdown, Labeling, Control, Membrane, Staining

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: Membranes were blocked in 5% (w/v) non-fat milk in TBST for 1 h, then incubated overnight at 4 °C with primary antibodies targeting PSMD14 (4197S; 1:1,000; Cell Signaling Technology), IMPDH2 (36281S; 1:1,000; Cell Signaling Technology), β-Actin (4970S; 1:1,000; Cell Signaling Technology), ubiquitin (20326S; 1:1,000; Cell Signaling Technology), K48-linkage-specific polyubiquitin (8081S; 1:1,000; Cell Signaling Technology), K63-linkage-specific polyubiquitin (5621S; 1:1,000; Cell Signaling Technology), His-Tag (12698T; 1:1000; Cell Signaling Technology), HA-Tag (3724S; 1:1000; Cell Signaling Technology) and Myc-Tag (2276S; 1:1,000; Cell Signaling Technology).

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