Journal: Nature Communications

Article Title: Competitive binding of E3 ligases TRIM26 and WWP2 controls SOX2 in glioblastoma

doi: 10.1038/s41467-021-26653-6

Figure Lengend Snippet: A GSC lysates treated with 100 μM cycloheximide (CHX) were subjected to immunoblotting. Data were representative of three independent experiments for B67, B36, and B66 and of two for BT87. B Quantification of protein expression levels for cycloheximide chase experiments in ( A ). Data represent mean ± SEM ( n = 3 for B67, B36, and B66. n = 2 for BT87. Unpaired t -test, ** P < 0.005 compared to 0 h). See Source Data File for exact P values and statistical parameters. C GSCs were treated with vehicle (DMSO) or 10 μM MG132 for 8 h. Lysates were subjected to immunoblotting. Data were representative of three independent experiments. D Schematic workflow for SOX2 immunoprecipitation from B36 GSC lysates followed by mass spectrometry (SOX2 IP-MS) to identify SOX2 interactors. E SOX2 IP-MS. (Left) 734 SOX2 interactors were identified. (Right) KEGG analysis for interactors was performed using Enrichr . F HEK293T cells were transfected with indicated DNA plasmids. Lysates were subjected to anti-GFP immunoprecipitation (IP) and immunoblotting (IB). Data were representative of three independent experiments. G GSC lysates were subjected to anti-SOX2 immunoprecipitation. Input lysates and IP samples were subjected to immunoblotting. Data were representative of three independent experiments. H Box plots for TRIM26 mRNA expression in TCGA IDH1/2 wild-type glioblastoma ( n = 142) and normal brain tissue ( n = 4) based on RNA-seq platform (Unpaired t -test, P = 0.0076). The boxes represent the 25th percentile, median, and 75th percentile values. The whiskers represent the maximum and minimum values for the plotted data See also Supplementary Fig. . See Source Data File for statistical parameters. I Patient tumors were subjected to single-cell RNA-sequencing (scRNA-seq). Malignant cells were identified by expression-based inference of copy number alterations. Cells are colored by expression levels for TRIM26 (left) and SOX2 (right). See also Supplementary Fig. . J An independent scRNA-seq dataset (Neftel et al, Cell , 2019) was examined for TRIM26 expression in 28 glioblastomas and plotted as a violin plot. Smart-seq2 data were downloaded with cell type annotations. (Malignant: n = 6863, Macrophage: n = 754, Oligodendrocyte: n = 219, T-cell: n = 94; two-sided t -test (malignant vs. nonmalignant), P = 0.002662).

Article Snippet: Lentiviral expression constructs were generated by cloning the following cDNAs into the pLV-EF1a-IRES-Blast plasmid (Addgene 85133): TRIM26 (WTres, I18Ares, and C16AC36Ares), GFP-TRIM26 PRYSPRY, WWP2res, SOX2-6X-His, and Renilla luciferase.

Techniques: Western Blot, Expressing, Immunoprecipitation, Mass Spectrometry, Protein-Protein interactions, Transfection, RNA Sequencing

Journal: Nature Communications

Article Title: Competitive binding of E3 ligases TRIM26 and WWP2 controls SOX2 in glioblastoma

doi: 10.1038/s41467-021-26653-6

Figure Lengend Snippet: A GSCs were transduced with TRIM26 RNAi or control. Five days later, lysates were analyzed by immunoblotting. Data were representative of three independent experiments. B GSCs transduced with TRIM26 RNAi or control were transduced with either vector, wild-type (WT) TRIM26res, or RING domain-mutant TRIM26res (I18A and C16AC36A) lentiviruses. Four days later, lysates were subjected to immunoblotting. Data were representative of three independent experiments. C GSCs transduced with TRIM26 RNAi or control were treated 5 days later with vehicle (DMSO) or 10 μM MG132 for 8 h. Lysates were subjected to immunoblotting. Data were representative of three independent experiments. D GSCs stably expressing histidine-tagged SOX2 and FLAG-tagged ubiquitin were transduced with TRIM26 RNAi or control. Lysates were subjected to immunoprecipitation (IP) under denaturing conditions using nickel-NTA beads. IP and input samples were analyzed by immunoblotting. Data were representative of three independent experiments. E GSCs transduced with Firefly luciferase-based SOX2 reporter and Renilla luciferase lentiviruses were transduced with TRIM26 RNAi or control. Five days later, luciferase activities were measured. Firefly activity was divided by Renilla activity for normalization. Data represent mean ± SEM ( n = 3, ANOVA, ** P < 0.01). See Source Data File for exact P values and statistical parameters. See also Supplementary Fig. . F GSCs stably transduced with luciferase-based SOX2 reporter lentiviruses were measured as in ( E ). Luciferase values were divided by protein concentration for normalization. Data represent mean ± SEM ( n = 3, ANOVA, * P < 0.03). See Source Data File for exact P values and statistical parameters. G Correlation analysis for TRIM26 and SOX2 mRNA in TCGA glioblastoma RNA-seq dataset ( n = 141, Pearson r = 0.59, P < 0.0001). H An independent scRNA-seq dataset (10X data, Neftel et al, Cell , 2019) was used to derive pseudobulk expression values for TRIM26 and SOX2 only in tumor cells for correlation analysis ( n = 9 tumors, linear regression, R 2 = 0.69, P = 0.00330). See also Supplementary Fig. . I B67 GSCs transduced with TRIM26 RNAi or control were harvested for total RNA and subjected to RNA-seq. The top 141 significantly differentially expressed (compared to control), directionally consistent genes between the two TRIM26 RNAi are plotted as a heatmap ( n = 3 per condition, FDR <0.05). See Source Data for data points. See also Supplementary Table . J Gene Ontology (GO) analysis was performed using significantly downregulated genes (FDR <0.05) shared by both TRIM26 RNAi (compared to control) from ( I ) using Enrichr . The top ten GO terms related to the biological process are shown. K Chromatin-immunoprecipitation-X (ChIP-X) enrichment analysis was performed using all significantly differentially regulated genes (FDR <0.05) shared by both TRIM26 RNAi from ( I ) using X2K . The top ten transcription factors with enriched target genes are shown. L Significantly differentially regulated (FDR <0.05) and directionally consistent genes shared by both TRIM26 RNAi (compared to control) were analyzed for expression of 27 GSC marker genes, and significant genes (FDR <0.05) shown by heatmap. See Source Data for data points. See also Supplementary Fig. .

Article Snippet: Lentiviral expression constructs were generated by cloning the following cDNAs into the pLV-EF1a-IRES-Blast plasmid (Addgene 85133): TRIM26 (WTres, I18Ares, and C16AC36Ares), GFP-TRIM26 PRYSPRY, WWP2res, SOX2-6X-His, and Renilla luciferase.

Techniques: Transduction, Control, Western Blot, Plasmid Preparation, Mutagenesis, Stable Transfection, Expressing, Ubiquitin Proteomics, Immunoprecipitation, Luciferase, Activity Assay, Protein Concentration, RNA Sequencing, Chromatin Immunoprecipitation, Marker

Journal: Nature Communications

Article Title: Competitive binding of E3 ligases TRIM26 and WWP2 controls SOX2 in glioblastoma

doi: 10.1038/s41467-021-26653-6

Figure Lengend Snippet: A GSCs were transduced with TRIM26 RNAi or control and subjected to the extreme limiting dilution assay (ELDA). Data represent mean ± SEM ( n = 3, ANOVA, *** P < 0.005). See Source Data File for exact P values and statistical parameters. B GSCs treated as in ( A ) were subjected to ELDA. Data represent mean ± SEM ( n = 5, ANOVA, *** P < 0.0001). See Source Data File for exact P values and statistical parameters. C GSCs transduced with TRIM26 RNAi or control were transduced 2 days later with SOX2-expressing or vector (Vec) lentiviruses and subjected to ELDA. Data represent mean ± SEM ( n = 3, ANOVA, * P < 0.05, *** P < 0.0005). See Source Data File for exact P values and statistical parameters. D In vitro translated, 35 S-labeled full-length (FL) TRIM26 and domain deletion mutants were used in GST pulldown assays using glutathione beads and bacterially produced GST control or GST-SOX2 (*FL SOX2). TRIM26 proteins were visualized by gel electrophoresis and fluorography (middle panel: input). GST-fusion proteins were visualized by Coomassie brilliant blue (CBB) staining. Data were representative of three independent experiments. E In vitro translated, 35 S-labeled full-length (FL) TRIM26, TRIM26 1-363, and TRIM26 PRYSPRY domain were used in GST pulldown assays with GST control or GST-SOX2 as in ( D ). Data were representative of three independent experiments. F GSCs transduced with TRIM26 RNAi or control were treated 2 days later with either vector or TRIM26 GFP-PRYSPRY-expressing lentiviruses. Four days later, lysates were subjected to immunoblotting. Data were representative of three independent experiments. G GSCs transduced with TRIM26 RNAi or control were transduced 2 days later with either vector (Vec) or TRIM26 GFP-PRYSPRY-expressing lentiviruses and then subjected to ELDA as in ( A ). Data represent mean ± SEM ( n = 5, ANOVA, ** P < 0.01, ns nonsignificant). See Source Data File for exact P values and statistical parameters.

Article Snippet: Lentiviral expression constructs were generated by cloning the following cDNAs into the pLV-EF1a-IRES-Blast plasmid (Addgene 85133): TRIM26 (WTres, I18Ares, and C16AC36Ares), GFP-TRIM26 PRYSPRY, WWP2res, SOX2-6X-His, and Renilla luciferase.

Techniques: Transduction, Control, Limiting Dilution Assay, Expressing, Plasmid Preparation, In Vitro, Labeling, Produced, Nucleic Acid Electrophoresis, Staining, Western Blot

Journal: Nature Communications

Article Title: Competitive binding of E3 ligases TRIM26 and WWP2 controls SOX2 in glioblastoma

doi: 10.1038/s41467-021-26653-6

Figure Lengend Snippet: A Transfected HEK293T cell lysates were subjected to immunoprecipitation (IP). Input lysates and IP samples were subjected to immunoblotting (IB). Data were representative of three independent experiments. B Transfected HEK293T cell lysates were subjected to immunoprecipitation (IP). Input lysates and IP samples were subjected to immunoblotting (IB). Data were representative of three independent experiments. C GSCs transduced with vector (Vec) or HA-TRIM26- or GFP-TRIM26 PRYSPRY-expressing lentiviruses were treated 4 days later with MG132 (10 μM) for 4 h to maintain SOX2 protein levels, and lysates were subjected to anti-SOX2 immunoprecipitation (IP). Input lysates and IP samples were subjected to immunoblotting. Data were representative of three independent experiments. D HEK293T cells were singly transfected with expression plasmids for HA-TRIM26 PRYSPRY, HA-WWP2, and GFP-SOX2, and lysates harvested. GFP-SOX2 and HA-WWP2 expressing lysates were added to all three conditions in equal amounts. HA-TRIM26 PRYSPRY-expressing lysate was excluded from the first condition and added in incremental amounts to the last two conditions. Lysates were subjected to anti-SOX2 immunoprecipitation (IP). IP samples were assayed by immunoblotting (IB). Data were representative of three independent experiments. E HEK293T cells singly transfected with HA-WWP2 and HA-TRIM26-I18A expression plasmids were harvested and subjected to anti-HA immunoprecipitation. Recombinant E1 and E2 (UBE2D3) enzymes were added to all three conditions. HA-WWP2 IP beads, HA-TRIM26-I18A IP beads, and in vitro synthesized, 35 S-labeled SOX2 were added as indicated. In vitro ubiquitination reactions were performed and subjected to electrophoresis and fluorography to detect SOX2. Data were representative of three independent experiments. F GSCs transduced with WWP2 RNAi or control were transduced with TRIM26 RNAi or control 2 days later. Four days later, lysates were subjected to immunoblotting. Data were representative of three independent experiments. G GSCs transduced with WWP2 RNAi or control were transduced with TRIM26 RNAi or control 2 days later and then subjected to ELDA as in Fig. . Data represent mean ± SEM ( n = 5, ANOVA, *** P < 0.0005, * P < 0.05, ns nonsignificant). See Source Data File for exact P values and additional statistical parameters.

Article Snippet: Lentiviral expression constructs were generated by cloning the following cDNAs into the pLV-EF1a-IRES-Blast plasmid (Addgene 85133): TRIM26 (WTres, I18Ares, and C16AC36Ares), GFP-TRIM26 PRYSPRY, WWP2res, SOX2-6X-His, and Renilla luciferase.

Techniques: Transfection, Immunoprecipitation, Western Blot, Transduction, Plasmid Preparation, Expressing, Recombinant, In Vitro, Synthesized, Labeling, Ubiquitin Proteomics, Electrophoresis, Control

Journal: Nature Communications

Article Title: Competitive binding of E3 ligases TRIM26 and WWP2 controls SOX2 in glioblastoma

doi: 10.1038/s41467-021-26653-6

Figure Lengend Snippet: A GSCs expressing Firefly luciferase were transduced with TRIM26 RNAi or control. Six days later, 2.5 × 10 5 GSCs were injected into the brains of mice. Bioluminescent live imaging (BLI) was performed 8 weeks post-injection. Four representative animals are shown. B Quantification of BLI in ( A ). Data represent mean ± SEM (Control: n = 12 for 4, 8 weeks; 6 for 12, 16 weeks. TRIM26i.1: n = 11 for 4, 8 weeks; 5 for 12, 16 weeks. TRIM26i.2: n = 13 for 4, 8 weeks; 7 for 12, 16 weeks. ANOVA, * P < 0.001 for RNAi compared to control, # P < 0.04 for TRIM26i.2 only). See Source Data File for exact P values and statistical parameters. C Neurological deficit-free survival of mice treated as in ( A ) is shown ( n = 6, log-rank, ** P < 0.001). See Source Data File for exact P values and statistical parameters. D About 1 × 10 4 GSCs transduced with TRIM26 RNAi or control were injected in mice as in ( A ). Neurological deficit-free survival is shown ( n = 5, log-rank, ** P < 0.005). See Source Data File for exact P values and statistical parameters. E About 5 × 10 4 MGG8 GSCs transduced with TRIM26 RNAi, WWP2 RNAi, or control were injected into mice. Four weeks later, brains were processed for human nuclear antigen (hNA) and SOX2 immunofluorescence. Bar = 50 μm. Data were representative of three independent experiments. F MGG8 GSCs transduced with WWP2 RNAi or control were injected into mice, and brains subjected to immunofluorescence as in ( E ). SOX2 intensity per cell nucleus was quantified by z-stack (Control: n = 225 nuclei; WWP2i.1: n = 293 nuclei in three animals per condition, unpaired t -test, P < 0.001). See Source Data File for exact P values and statistical parameters. G GSCs transduced with WWP2 RNAi or control were transduced 2 days later with TRIM26 RNAi or control. About 5 × 10 4 GSCs were injected into mice. Neurological deficit-free survival is shown ( n = 6 for WWP2i and n = 12 for Control, TRIM26i.2, and TRIM26i.2 + WWP2i.1. log-rank, ** P < 0.005, *** P < 0.001, **** P < 0.0001, ns nonsignificant). See Source Data File for exact P values and statistical parameters. H Schematic of TRIM26/WWP2 competitive binding model with SOX2 in GSCs.

Article Snippet: Lentiviral expression constructs were generated by cloning the following cDNAs into the pLV-EF1a-IRES-Blast plasmid (Addgene 85133): TRIM26 (WTres, I18Ares, and C16AC36Ares), GFP-TRIM26 PRYSPRY, WWP2res, SOX2-6X-His, and Renilla luciferase.

Techniques: Expressing, Luciferase, Transduction, Control, Injection, Imaging, Immunofluorescence, Binding Assay

Journal: Cell reports

Article Title: MMD collaborates with ACSL4 and MBOAT7 to promote polyunsaturated phosphatidylinositol remodeling and susceptibility to ferroptosis

doi: 10.1016/j.celrep.2023.113023

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: To re-express MMD in MMD knockout cells, the HA-MMD cDNA sequence was edited by fusion PCR (final sequence) to reduce base-pairing with both MMD-sg8 and MMD-sg10 and cloned into the lentiviral vector pLV-EF1a-IRES-blast by restriction digestion with EcoRI-HF (NEB) and BamHI-HF (NEB).

Techniques: Virus, Recombinant, Cell Viability Assay, Mass Spectrometry, Luciferase, CRISPR, Sequencing, Plasmid Preparation, Clone Assay, Expressing, Variant Assay, Software

Journal: Cell reports

Article Title: MMD collaborates with ACSL4 and MBOAT7 to promote polyunsaturated phosphatidylinositol remodeling and susceptibility to ferroptosis

doi: 10.1016/j.celrep.2023.113023

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: To re-express MMD in MMD knockout cells, the HA-MMD cDNA sequence was edited by fusion PCR (final sequence) to reduce base-pairing with both MMD-sg8 and MMD-sg10 and cloned into the lentiviral vector pLV-EF1a-IRES-blast by restriction digestion with EcoRI-HF (NEB) and BamHI-HF (NEB).

Techniques: Virus, Recombinant, Cell Viability Assay, Mass Spectrometry, Luciferase, CRISPR, Sequencing, Plasmid Preparation, Clone Assay, Expressing, Variant Assay, Software