Journal: Cell Death & Disease

Article Title: ZC3H15 regulates the ubiquitination of PTEN via recruitment of TRIM56 and promotes malignant progression of non-small cell lung cancer

doi: 10.1038/s41419-025-08138-2

Figure Lengend Snippet: A The Venn diagram shows the overlaps between protein mass spectrometry analysis results and the data from BioGRID website. B The raw MS/MS spectrum of PTEN. C Immunofluorescence assay results indicate that ZC3H15 and PTEN colocalize within the cytoplasm of A549 and H1299 cells. D , E Interactions between PHF23 and ACTN4 in A549 and H1299 cells measured by co-immunoprecipitation. F Cell growth of A549 treated with DMSO or VO-Ohpic was determined by colony formation. Unpaired t test. Mean ± SD, n = 3. *** P < 0.001. G DNA replication of A549 treated with DMSO or VO-Ohpic was determined by EDU staining. Scale bar: 20 μm. Mean ± SD, n = 3. ** P < 0.01, *** P < 0.001. H Cell migration and invasion of A549 was evaluated by the Transwell migration assay. Mean ± SD, n = 3. ** P < 0.01, *** P < 0.001. I The migration of A549 treated with DMSO or VO-Ohpic was evaluated by the wound-healing assay. Mean ± SD, n = 3. *** P < 0.001.

Article Snippet: For inhibitor assay, cells were treated with the AKT-mTOR pathway inhibitor LY294002 (40 μM; MedChemExpress, Monmouth Junction, NJ, USA) [ ] or the PTEN inhibitor VO-Ohpic trihydrate (50 nM; MedChemExpress) and incubated for 24 h. All inhibitor concentrations were selected based on established efficacy from previous studies and our preliminary dose-response experiments.

Techniques: Mass Spectrometry, Tandem Mass Spectroscopy, Immunofluorescence, Immunoprecipitation, Staining, Migration, Transwell Migration Assay, Wound Healing Assay

Journal: Cell Death & Disease

Article Title: ZC3H15 regulates the ubiquitination of PTEN via recruitment of TRIM56 and promotes malignant progression of non-small cell lung cancer

doi: 10.1038/s41419-025-08138-2

Figure Lengend Snippet: A Schematic diagram of ZC3H15 splicing mutants. B The expression of myc-tag after transfected with ZC3H15 splicing mutant cDNA. C Interactions between ZC3H15 splicing mutants and PTEN in A549 and H1299 cells measured by co-immunoprecipitation. D Cell viability of A549 and H1299 transfected with ZC3H15 cDNA or ZC3H15 MUT3 cDNA was analyzed by CCK8. Unpaired t test. Mean ± SD, n = 3. *** P < 0.001. E Cell growth of A549 transfected with ZC3H15 cDNA or ZC3H15 MUT3 cDNA was determined by colony formation. Mean ± SD, n = 3. *** P < 0.001. F DNA replication of A549 transfected with ZC3H15 cDNA or ZC3H15 MUT3 cDNA was determined by EDU staining. Scale bar: 20 μm. Mean ± SD, n = 3. *** P < 0.001. The migration ( G ) and invasion ( H ) of A549 transfected with ZC3H15 cDNA or ZC3H15 MUT3 cDNA was evaluated by the Transwell migration assay. Mean ± SD, n = 3. *** P < 0.001. I Western blotting analyzing the expression of proteins involved in the AKT-mTOR pathway and proliferation- and migration-related proteins in A549 and H1299 cells transfected with ZC3H15 cDNA or ZC3H15 MUT3 cDNA.

Article Snippet: For inhibitor assay, cells were treated with the AKT-mTOR pathway inhibitor LY294002 (40 μM; MedChemExpress, Monmouth Junction, NJ, USA) [ ] or the PTEN inhibitor VO-Ohpic trihydrate (50 nM; MedChemExpress) and incubated for 24 h. All inhibitor concentrations were selected based on established efficacy from previous studies and our preliminary dose-response experiments.

Techniques: Expressing, Transfection, Mutagenesis, Immunoprecipitation, Staining, Migration, Transwell Migration Assay, Western Blot

Journal: Cell Death & Disease

Article Title: ZC3H15 regulates the ubiquitination of PTEN via recruitment of TRIM56 and promotes malignant progression of non-small cell lung cancer

doi: 10.1038/s41419-025-08138-2

Figure Lengend Snippet: A Levels of PTEN ubiquitination were evaluated by immunoprecipitation using an anti-PTEN antibody, followed by anti-HA immunoblotting. B , C Levels of PTEN ubiquitination of A549 and H1299 transfected with K48/K63 mutant Ub were evaluated by immunoprecipitation using an anti-PTEN antibody, followed by anti-HA immunoblotting. D 3D Binding model analysis (ZC3H15 in pink, TRIM56 in blue and PTEN in cyan). The key residues are shown as sticks. H-bonds are shown as yellow dashed lines. E Confocal microscopy of triplestained Flag-ZC3H15(green), TRIM56(red) and DAPI(blue) in H1299 cells. F Confocal microscopy of triplestained GFP-PTEN(green), TRIM56(red) and DAPI(blue) in H1299 cells. G Interactions between TRIM56 with Flag-ZC3H15 and GFP-PTEN in A549 and H1299 cells measured by co-immunoprecipitation. H Levels of PTEN ubiquitination of A549 and H1299 with TRIM56 knockdown were evaluated by immunoprecipitation using an anti-PTEN antibody, followed by anti-HA immunoblotting. I Levels of PTEN ubiquitination were evaluated by immunoprecipitation using an anti-PTEN antibody, followed by anti-HA immunoblotting.

Article Snippet: For inhibitor assay, cells were treated with the AKT-mTOR pathway inhibitor LY294002 (40 μM; MedChemExpress, Monmouth Junction, NJ, USA) [ ] or the PTEN inhibitor VO-Ohpic trihydrate (50 nM; MedChemExpress) and incubated for 24 h. All inhibitor concentrations were selected based on established efficacy from previous studies and our preliminary dose-response experiments.

Techniques: Ubiquitin Proteomics, Immunoprecipitation, Western Blot, Transfection, Mutagenesis, Binding Assay, Confocal Microscopy, Knockdown

Journal: Cell Death & Disease

Article Title: ZC3H15 regulates the ubiquitination of PTEN via recruitment of TRIM56 and promotes malignant progression of non-small cell lung cancer

doi: 10.1038/s41419-025-08138-2

Figure Lengend Snippet: A IC50 analysis of NSCLC patients with high and low ZC3H15 expression based on TCGA data. B Western blotting analyzing the expression of PTEN and p-PTEN in A549 and A549-DDP. C Viability of A549 and A549-DDP cells was analyzed by CCK8 24 h after treatment with different concentrations of cisplatin. D Viability of A549 and H1299 cells with ZC3H15 overexpressed was analyzed by CCK8 24 h after treatment with different concentrations of cisplatin. E Representative explanted tumor growth curve of mice treated as indicated ( n = 5 per group). F Xenograft tumors from H1299 cells. G Tumor growth curves were shown. Mean ± SD, n = 5. *** P < 0.001. H Quantification of xenograft tumor weights. Mean ± SD, n = 5. *** P < 0.001. I Representative pictures of H&E and IHC staining of ZC3H15, Ki-67, and PTEN in the indicated xenograft tumors. Scale bar, 200 μm.

Article Snippet: For inhibitor assay, cells were treated with the AKT-mTOR pathway inhibitor LY294002 (40 μM; MedChemExpress, Monmouth Junction, NJ, USA) [ ] or the PTEN inhibitor VO-Ohpic trihydrate (50 nM; MedChemExpress) and incubated for 24 h. All inhibitor concentrations were selected based on established efficacy from previous studies and our preliminary dose-response experiments.

Techniques: Expressing, Western Blot, Immunohistochemistry

Journal: Cell Death & Disease

Article Title: ZC3H15 regulates the ubiquitination of PTEN via recruitment of TRIM56 and promotes malignant progression of non-small cell lung cancer

doi: 10.1038/s41419-025-08138-2

Figure Lengend Snippet: ZC3H15 promotes the proliferation, migration, invasion and chemotherapy resistance of NSCLC by mediating PTEN ubiquitination degradation and activating the AKT-mTOR signaling pathway.

Article Snippet: For inhibitor assay, cells were treated with the AKT-mTOR pathway inhibitor LY294002 (40 μM; MedChemExpress, Monmouth Junction, NJ, USA) [ ] or the PTEN inhibitor VO-Ohpic trihydrate (50 nM; MedChemExpress) and incubated for 24 h. All inhibitor concentrations were selected based on established efficacy from previous studies and our preliminary dose-response experiments.

Techniques: Migration, Ubiquitin Proteomics

Journal: International Journal of Molecular Medicine

Article Title: FBXO22 promotes hepatocellular carcinoma progression via paracrine myo-inositol-induced M2-type polarization of macrophages

doi: 10.3892/ijmm.2025.5707

Figure Lengend Snippet: FBXO22 induces PTEN ubiquitination and subsequent degradation in THP-1 cells. 97H cells were transfected with oe- FBXO22 . Reverse transcription-quantitative PCR and western blotting were performed to detect mRNA expression levels and protein levels of (A, C and D) FBXO22 , (B, C and E) IMPA1 and (G and H) PTEN . (F) A myo-inositol detection kit was used to detect myo-inositol in cell supernatants. (I and J) 97H cells were transfected with oe- FBXO22 , followed by the treatment with CHX (200 μ g/ml) for different periods (2, 4 and 8 h). Western blotting was performed to detect PTEN protein levels. (K) 97H cells were transfected with oe- FBXO22 followed by the treatment with MG132 (10 μ M) for 12 h. Western blotting was performed to detect PTEN protein levels. (L) 97H cells were incubated with an anti-PTEN antibody, and western blotting was performed to detect FBXO22 protein levels. (M) 97H cells were incubated with an anti-PTEN antibody, followed by the transfection with oe- FBXO22 . Western blotting was performed to detect PTEN ubiquitination levels. * P<0.05, ** P<0.01 and *** P<0.001. CHX, cycloheximide; oe-, overexpression; NC, negative control.

Article Snippet: The membrane was maintained with 5% non-fat milk for 1 h at room temperature, and incubated with the primary antibodies against the following proteins: PI3K (1:10,000; cat. no. 67071-1-Ig; Proteintech Group, Inc.), phosphorylated (p-) PI3K (1:1,000; cat. no. AF3242; Affinity Biosciences), AKT1 (1:10,000; cat. no. 80457-1-RR; Proteintech Group, Inc.), p-AKT1 (1:5,000; cat. no. 80462-1-RR, Proteintech Group, Inc.), FBXO22 (1:2,000; cat. no. 13606-1-AP; Proteintech Group, Inc.), IMPA1 (1:300; cat. no. 16593-1-AP; Proteintech Group, Inc.), SLC5A3 (1:1,000; cat. no. DF4521; Affinity Biosciences), PTEN (1:5,000; cat. no. 60300-1-Ig; Proteintech Group, Inc.), and NRF2 (1:2,000; cat. no. 16396-1-AP; Proteintech Group, Inc.). β-actin protein level was the internal control.

Techniques: Ubiquitin Proteomics, Transfection, Reverse Transcription, Real-time Polymerase Chain Reaction, Western Blot, Expressing, Incubation, Over Expression, Negative Control

Journal: International Journal of Molecular Medicine

Article Title: FBXO22 promotes hepatocellular carcinoma progression via paracrine myo-inositol-induced M2-type polarization of macrophages

doi: 10.3892/ijmm.2025.5707

Figure Lengend Snippet: FBXO22-induced myo-inositol release promotes M2 polarization via the regulation of the NRF2/IMPA1 axis in THP-1 cells. (A) 97H cells were incubated with an anti-NRF2 antibody, and western blotting was performed to detect PTEN protein levels. (B) 97H cells were transfected with oe- FBXO22 , and western blotting was performed to detect NRF2 protein level. (C) 293T cells were transfected with oe- NRF2 , and luciferase assay was performed to determine the interaction between NRF2 and IMPA1. (D) Chromatin immunoprecipitation assay was used to detect the transcriptional regulation of IMPA1 by NRF2 in 97H cells transfected with oe- FBXO22 , IgG was used as negative control. 97H cells were co-transfected with oe- FBXO22 and si- IMPA1 . (E and F) Reverse transcription-quantitative PCR and western blotting were performed to detect IMPA1 expression. (G) A myo-inositol detect kit was used to detect myo-inositol in cell supernatants. (H and I) Flow cytometry was performed to detect CD86-positive and CD206-positive THP-1 cells. ** P<0.01 and *** P<0.001. oe-, overexpression; si-, small interfering; NC, negative control.

Article Snippet: The membrane was maintained with 5% non-fat milk for 1 h at room temperature, and incubated with the primary antibodies against the following proteins: PI3K (1:10,000; cat. no. 67071-1-Ig; Proteintech Group, Inc.), phosphorylated (p-) PI3K (1:1,000; cat. no. AF3242; Affinity Biosciences), AKT1 (1:10,000; cat. no. 80457-1-RR; Proteintech Group, Inc.), p-AKT1 (1:5,000; cat. no. 80462-1-RR, Proteintech Group, Inc.), FBXO22 (1:2,000; cat. no. 13606-1-AP; Proteintech Group, Inc.), IMPA1 (1:300; cat. no. 16593-1-AP; Proteintech Group, Inc.), SLC5A3 (1:1,000; cat. no. DF4521; Affinity Biosciences), PTEN (1:5,000; cat. no. 60300-1-Ig; Proteintech Group, Inc.), and NRF2 (1:2,000; cat. no. 16396-1-AP; Proteintech Group, Inc.). β-actin protein level was the internal control.

Techniques: Incubation, Western Blot, Transfection, Luciferase, Chromatin Immunoprecipitation, Negative Control, Reverse Transcription, Real-time Polymerase Chain Reaction, Expressing, Flow Cytometry, Over Expression

Journal: Molecular Therapy. Nucleic Acids

Article Title: Systemic miR-26a deficiency attenuates pulmonary fibrosis via PTEN upregulation and downstream TIMP-1 suppression

doi: 10.1016/j.omtn.2025.102765

Figure Lengend Snippet: PTEN as a direct target of miR-26a (A) Sequence alignment showing the complementarity between miR-26a and PTEN. (B) Quantitative PCR analysis of Pten mRNA expression in WT and miR-26a KO murine lungs on day 7 after BLM administration ( n = 5/group). (C) ELISA analysis of PTEN protein levels in WT and miR-26a KO murine lungs on day 14 after BLM administration ( n = 7–8/group). (D) Immunohistochemical staining of WT and miR-26a KO murine lungs on day 14 after BLM administration using anti-PTEN antibodies. Scale bars, 50 μm. (E) Quantification of the stained area (% of total field) in the immunohistochemical staining images shown in D ( n = 4/group).

Article Snippet: The expressions of mRNA were measured using quantitative PCR with the Applied Biosystems 7500 Fast Real-Time PCR System (Applied Biosystems) with the following TaqMan Gene Expression Assays (all from Applied Biosystems) according to the manufacturer’s protocol: Tgfb1 (Assay ID, Mm03024053_m1), Il6 (Assay ID, Mm00446190_m1), Pten (Assay ID, Mm00477208_m1), Timp1 (Assay ID, Mm01341361_m1), Col1a1 (Assay ID, Mm00801666_g1), Acta2 (Assay ID, Mm00725412_s1), Pten (Assay ID, Hs02621230_s1), Timp1 (Assay ID, Hs01092512_g1), and Acta2 (Assay ID, Hs00426835_g1).

Techniques: Sequencing, Real-time Polymerase Chain Reaction, Expressing, Enzyme-linked Immunosorbent Assay, Immunohistochemical staining, Staining

Journal: Molecular Therapy. Nucleic Acids

Article Title: Systemic miR-26a deficiency attenuates pulmonary fibrosis via PTEN upregulation and downstream TIMP-1 suppression

doi: 10.1016/j.omtn.2025.102765

Figure Lengend Snippet: Differences in the effects of miR-26a in mouse-derived cells (A, E, I) miR-26a levels in primary lung fibroblasts transfected with control, siRNA control, and miR-26a. (A) shows fibroblasts derived from WT mice; (E) shows those from miR-26a KO mice; (I) shows LA-4 cells ( n = 4/group). (B–D) Relative changes in Pten , Timp1 , and Acta2 mRNA expression levels in primary lung fibroblasts derived from WT mice after miR-26a transfection, analyzed using quantitative PCR ( n = 4/group). (F–H) Relative changes in Pten , Timp1 , and Acta2 mRNA expression levels in primary lung fibroblasts derived from miR-26a KO mice after miR-26a transfection, analyzed using quantitative PCR ( n = 4/group). (J–L) Relative changes in Pten , Timp1 , and Acta2 mRNA expression levels in LA-4 cells after miR-26a transfection, analyzed using quantitative PCR ( n = 4/group).

Article Snippet: The expressions of mRNA were measured using quantitative PCR with the Applied Biosystems 7500 Fast Real-Time PCR System (Applied Biosystems) with the following TaqMan Gene Expression Assays (all from Applied Biosystems) according to the manufacturer’s protocol: Tgfb1 (Assay ID, Mm03024053_m1), Il6 (Assay ID, Mm00446190_m1), Pten (Assay ID, Mm00477208_m1), Timp1 (Assay ID, Mm01341361_m1), Col1a1 (Assay ID, Mm00801666_g1), Acta2 (Assay ID, Mm00725412_s1), Pten (Assay ID, Hs02621230_s1), Timp1 (Assay ID, Hs01092512_g1), and Acta2 (Assay ID, Hs00426835_g1).

Techniques: Derivative Assay, Transfection, Control, Expressing, Real-time Polymerase Chain Reaction

Journal: Molecular Therapy. Nucleic Acids

Article Title: Systemic miR-26a deficiency attenuates pulmonary fibrosis via PTEN upregulation and downstream TIMP-1 suppression

doi: 10.1016/j.omtn.2025.102765

Figure Lengend Snippet: Differences in the effects of miR-26a in human-derived cells (A and E) miR-26a levels in MRC-5 cells and A549 cells transfected with control, siRNA control, and miR-26a. (A) shows MRC-5 cells; (E) shows A549 cells ( n = 4/group). (B–D) Relative changes in Pten , Timp1 , and Acta2 mRNA expression levels in MRC-5 cells after miR-26a transfection, analyzed using quantitative PCR ( n = 4/group). (F–H) Relative changes in Pten , Timp1 , and Acta2 mRNA expression levels in A549 cells after miR-26a transfection, analyzed using quantitative PCR ( n = 4/group).

Article Snippet: The expressions of mRNA were measured using quantitative PCR with the Applied Biosystems 7500 Fast Real-Time PCR System (Applied Biosystems) with the following TaqMan Gene Expression Assays (all from Applied Biosystems) according to the manufacturer’s protocol: Tgfb1 (Assay ID, Mm03024053_m1), Il6 (Assay ID, Mm00446190_m1), Pten (Assay ID, Mm00477208_m1), Timp1 (Assay ID, Mm01341361_m1), Col1a1 (Assay ID, Mm00801666_g1), Acta2 (Assay ID, Mm00725412_s1), Pten (Assay ID, Hs02621230_s1), Timp1 (Assay ID, Hs01092512_g1), and Acta2 (Assay ID, Hs00426835_g1).

Techniques: Derivative Assay, Transfection, Control, Expressing, Real-time Polymerase Chain Reaction