Journal: Journal of Translational Medicine

Article Title: Hypoxia-induced PRMT6 expression promotes temozolomide chemoresistance in glioblastoma via G3BP1

doi: 10.1186/s12967-025-07618-5

Figure Lengend Snippet: PRMT6 is highly expressed in gliomas and correlates with poor prognosis. ( A ) Pan-cancer analysis of PRMT6 in the TCGA and GTEx databases shows that PRMT6 is generally highly expressed in tumors compared to normal tissues, with a significant difference in glioma. ( B ) Univariate Log-rank test of pan-cancer survival data in the TCGA dataset indicates that PRMT6 is associated with poor prognosis in glioma. ( C ) PRMT6 expression in different WHO grades of glioma in the TCGA and CGGA datasets. ( D ) PRMT6 expression in glioma subtypes in the CGGA dataset. ( E ) Protein expression of PRMT6 in different grades of glioma patients and non-tumor tissues. ( F ) Representative immunohistochemical images of PRMT6 in different grades of glioma samples and non-tumor tissues. Scale bar = 100 μm. ( G ) ROC curves for 1-year, 3-year, and 5-year survival of glioma patients based on PRMT6 expression in the TCGA and CGGA datasets. ( H ) Kaplan-Meier survival analysis shows the overall survival of glioma patients with high and low PRMT6 expression in the TCGA and CGGA datasets. ( I ) Kaplan-Meier survival analysis of overall survival in glioma patients treated with temozolomide, stratified by high and low PRMT6 expression in the CGGA dataset. Data in A-D are presented as mean ± SD. For E and F, n = 3 biologically independent samples. Statistical significance in A, C, and D was assessed by one-way ANOVA followed by Tukey’s test for multiple comparisons. The significance of data in B, H, and I was assessed using the Log-rank test for survival comparisons. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: Antibodies included: Anti-PRMT6 antibody (Cat No. 14641) and anti-ADMA antibody (Cat No. 13522) (CST; Boston, Massachusetts, USA), anti-HIF-1α (Cat No. 20960-1-AP), anti-G3BP1 (Cat No. 13057-2-AP), anti-GABPA (Cat No. 21542-1-AP), and anti-BCL2L13 (Cat No. 16612-1-AP) from Proteintech (Wuhan, China); and anti-GAPDH (Cat No. bs-2188R) from Bioss (Beijing, China).

Techniques: Expressing, Immunohistochemical staining

Journal: Journal of Translational Medicine

Article Title: Hypoxia-induced PRMT6 expression promotes temozolomide chemoresistance in glioblastoma via G3BP1

doi: 10.1186/s12967-025-07618-5

Figure Lengend Snippet: PRMT6 promotes glioblastoma proliferation and temozolomide resistance. ( A ) Verification of PRMT6 protein expression after PRMT6 knockdown in U251 and LN229 cell lines. ( B ) Verification of PRMT6 mRNA expression after PRMT6 knockdown in U251 and LN229 cell lines ( C ) Colony formation assay showing the impact of PRMT6 knockdown on cell proliferation (treated with 100µM TMZ or DMSO) in U251 and LN229 cell lines. ( D ) CCK-8 assay showing the impact of PRMT6 knockdown on cell proliferation (treated with 100µM TMZ or DMSO) in U251 and LN229 cell lines. ( E ) Flow cytometric analysis of cell cycle progression and proliferation in PRMT6-knockdown cells (treated with 100µM TMZ or DMSO). ( F ) Comparative tumor sphere size analysis of PRMT6-knockdown U251 and LN229 cells after 24 h culture with 100µM TMZ. Scale bar = 500 μm. ( G ) Decreased TMZ IC50 values in PRMT6-knockdown cells indicate enhanced TMZ sensitivity. ( H ) Flow cytometric apoptosis profiles of PRMT6-knockdown U251 and LN229 cells treated with 200µM TMZ. ( I ) Quantitative apoptosis ratios in PRMT6-knockdown cells following 200µM TMZ treatment. Data in B-G, and I are presented as mean ± SD. For A-H, n = 3 biologically independent samples. Statistical significance in B-G, and I was assessed by one-way ANOVA followed by Tukey’s test for multiple comparisons. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: Antibodies included: Anti-PRMT6 antibody (Cat No. 14641) and anti-ADMA antibody (Cat No. 13522) (CST; Boston, Massachusetts, USA), anti-HIF-1α (Cat No. 20960-1-AP), anti-G3BP1 (Cat No. 13057-2-AP), anti-GABPA (Cat No. 21542-1-AP), and anti-BCL2L13 (Cat No. 16612-1-AP) from Proteintech (Wuhan, China); and anti-GAPDH (Cat No. bs-2188R) from Bioss (Beijing, China).

Techniques: Expressing, Knockdown, Colony Assay, CCK-8 Assay

Journal: Journal of Translational Medicine

Article Title: Hypoxia-induced PRMT6 expression promotes temozolomide chemoresistance in glioblastoma via G3BP1

doi: 10.1186/s12967-025-07618-5

Figure Lengend Snippet: HIF-1α associates with PRMT6 and regulates its transcription. ( A ) Correlation analysis of HIF-1α and PRMT6 expression in gliomas using mRNA sequencing data from TCGA and CGGA databases. ( B ) Time-course analysis of HIF-1α, PRMT6, and G3BP1 protein expression in LN229 and U251 cells under hypoxic conditions. ( C ) HIF-1α, PRMT6 and G3BP1 protein expression in LN229 and U251 cells treated with CoCl2 concentration gradient. ( D ) Immunofluorescence analysis of HIF-1α (red) and PRMT6 (green) in HIF-1α-knockdown cells under normoxia or hypoxia conditions. Scale bar = 100 μm. ( E ) HIF-1α, PRMT6 and G3BP1 protein expression in HIF-1α-knockdown cells under normoxia or hypoxia conditions. ( F ) HIF-1α and PRMT6 mRNA expression in knockdown cells under normoxic or hypoxic conditions. ( G ) Five predicted HIF-1α binding sites in PRMT6 promoter region. ( H ) ChIP-qPCR analysis of HIF-1α binding at predicted sites in U251 and LN229 cells, showing strongest binding at HRE3. ( I ) Dual-luciferase reporter assay of HRE3-driven PRMT6 promoter activity in HIF-1α-knockdown U251 and LN229 cells under normoxic or hypoxic conditions. Data in B-F, H, and I are presented as mean ± SD. For B-F, H, and I, n = 3 biologically independent samples. Statistical significance in B-F, H, and I was assessed by one-way ANOVA followed by Tukey’s test for multiple comparisons: * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: Antibodies included: Anti-PRMT6 antibody (Cat No. 14641) and anti-ADMA antibody (Cat No. 13522) (CST; Boston, Massachusetts, USA), anti-HIF-1α (Cat No. 20960-1-AP), anti-G3BP1 (Cat No. 13057-2-AP), anti-GABPA (Cat No. 21542-1-AP), and anti-BCL2L13 (Cat No. 16612-1-AP) from Proteintech (Wuhan, China); and anti-GAPDH (Cat No. bs-2188R) from Bioss (Beijing, China).

Techniques: Expressing, Sequencing, Concentration Assay, Immunofluorescence, Knockdown, Binding Assay, ChIP-qPCR, Luciferase, Reporter Assay, Activity Assay

Journal: Journal of Translational Medicine

Article Title: Hypoxia-induced PRMT6 expression promotes temozolomide chemoresistance in glioblastoma via G3BP1

doi: 10.1186/s12967-025-07618-5

Figure Lengend Snippet: Transcriptomic analysis identifies G3BP1 as the key mediator of PRMT6-induced temozolomide resistance. ( A ) Comparative TMZ IC50 values between isogenic wild-type and resistant variants of U251 and LN229 cell lines. ( B ) Flow cytometric apoptosis profiles of isogenic wild-type and resistant cell lines treated with 200µM TMZ. ( C ) Quantitative apoptosis ratios in wild-type versus resistant variants following 200µM TMZ treatment. ( D ) Colony formation assay of isogenic cell pairs under TMZ concentration gradients. ( E ) Tumor sphere size comparison between wild-type and resistant variants cultured with 100µM TMZ for 24 h. Scale bar = 500 μm. ( F ) Volcano plot of differential gene expression in PRMT6-knockdown versus control U251 or LN229 cell lines, identifying PRMT6 and G3BP1 as significantly downregulated genes. ( G ) Volcano plot of U251 TMZ-resistant versus wild-type cells, showing PRMT6 and G3BP1 as prominently upregulated genes in resistant variants. ( H ) Venn diagram intersection of differentially expressed genes from PRMT6-knockdown cells and TMZ-resistant variants, highlighting G3BP1 among 23 overlapping genes with strongest PRMT6 correlation. ( I ) Heatmap visualization of the 23 intersecting genes’ expression patterns in resistant versus wild-type cell pairs. ( J ) Western blot analysis of PRMT6 and G3BP1 protein expression in U251 wild-type and its isogenic TMZ-resistant counterpart. ( K ) Immunofluorescence analysis of PRMT6 (green) and G3BP1 (red) in wild-type and resistant variants. Scale bar = 100 μm. Data in A, C, D, E, J, and K are presented as mean ± SD. For A, B, D, E, F, G, J, and K, n = 3 biologically independent samples. Statistical significance in A, C, D, E, J, and K was assessed by one-way ANOVA followed by Tukey’s test for multiple comparisons. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Source data are provided as source data files

Article Snippet: Antibodies included: Anti-PRMT6 antibody (Cat No. 14641) and anti-ADMA antibody (Cat No. 13522) (CST; Boston, Massachusetts, USA), anti-HIF-1α (Cat No. 20960-1-AP), anti-G3BP1 (Cat No. 13057-2-AP), anti-GABPA (Cat No. 21542-1-AP), and anti-BCL2L13 (Cat No. 16612-1-AP) from Proteintech (Wuhan, China); and anti-GAPDH (Cat No. bs-2188R) from Bioss (Beijing, China).

Techniques: Colony Assay, Concentration Assay, Comparison, Cell Culture, Gene Expression, Knockdown, Control, Expressing, Western Blot, Immunofluorescence

Journal: Journal of Translational Medicine

Article Title: Hypoxia-induced PRMT6 expression promotes temozolomide chemoresistance in glioblastoma via G3BP1

doi: 10.1186/s12967-025-07618-5

Figure Lengend Snippet: PRMT6 interacts with GABPA to transcriptionally regulate G3BP1 expression. ( A ) Correlation analysis of PRMT6 and G3BP1 expression in gliomas using mRNA sequencing data from TCGA and CGGA databases. ( B ) PRMT6 and G3BP1 protein expression in U251 and LN229 cell lines following PRMT6 knockdown. ( C ) PRMT6 and G3BP1 mRNA expression in U251 and LN229 cells lines after PRMT6 knockdown. ( D ) Immunofluorescence analysis of PRMT6 (green) and G3BP1 (red) in PRMT6-knockdown U251 and LN229 cells. Scale bar = 100 μm. ( E ) Venn diagram showing overlap between PRMT6-interacting proteins identified by mass spectrometry in LN229 cells and G3BP1-regulating transcription factors from Cistrome and KnockTF databases. ( F ) AlphaFold3-predicted molecular docking model of PRMT6 and GABPA proteins. ( G ) Co-immunoprecipitation (Co-IP) assay demonstrating PRMT6-GABPA interaction in U251 and LN229 cells. ( H ) Fluorescence co-localization of PRMT6 (green) and GABPA (red). Scale bar = 10 μm. ( I ) Eight predicted GABPA binding sites in the G3BP1 promoter region. ( J ) ChIP-qPCR analysis of PRMT6 and GABPA binding at predicted sites in U251 and LN229 cells, showing strongest binding at HRE5. ( K ) Dual-luciferase reporter assay measuring HRE5-driven G3BP1 promoter activity in GABPA-knockdown versus control U251 and LN229 cells. ( L ) ChIP-qPCR analysis of PRMT6 and GABPA binding to G3BP1 HRE5 in GABPA-knockdown and control cells. ( M ) Dual-luciferase reporter assay in HEK-293T cells under PRMT6 knockout conditions to evaluate luciferase activity in GABPA-knockdown versus control groups. ( N ) G3BP1 protein expression in HEK-293T cells with combined PRMT6 knockdown and GABPA knockdown. Data in B-D, and J-M are presented as mean ± SD. For B-D, G, H, and J-N, n = 3 biologically independent samples. Statistical significance in B-D, and J-M was assessed by one-way ANOVA followed by Tukey’s test for multiple comparisons. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: Antibodies included: Anti-PRMT6 antibody (Cat No. 14641) and anti-ADMA antibody (Cat No. 13522) (CST; Boston, Massachusetts, USA), anti-HIF-1α (Cat No. 20960-1-AP), anti-G3BP1 (Cat No. 13057-2-AP), anti-GABPA (Cat No. 21542-1-AP), and anti-BCL2L13 (Cat No. 16612-1-AP) from Proteintech (Wuhan, China); and anti-GAPDH (Cat No. bs-2188R) from Bioss (Beijing, China).

Techniques: Expressing, Sequencing, Knockdown, Immunofluorescence, Mass Spectrometry, Co-Immunoprecipitation Assay, Fluorescence, Binding Assay, ChIP-qPCR, Luciferase, Reporter Assay, Activity Assay, Control, Knock-Out

Journal: Journal of Translational Medicine

Article Title: Hypoxia-induced PRMT6 expression promotes temozolomide chemoresistance in glioblastoma via G3BP1

doi: 10.1186/s12967-025-07618-5

Figure Lengend Snippet: Hypoxia promotes TMZ resistance in glioblastoma through the HIF-1α-PRMT6-G3BP1 axis. ( A ) G3BP1 protein expression in LN229 cells with PRMT6 knockdown versus controls under normoxic or hypoxic conditions, demonstrating that hypoxia-mediated regulation of G3BP1 is less pronounced than PRMT6-mediated regulation. ( B ) TMZ IC50 values in PRMT6-knockdown and control LN229 cells under different oxygen conditions. ( C ) Flow cytometric apoptosis profiles of PRMT6-knockdown and control LN229 cells treated with 200µM TMZ under normoxic or hypoxic conditions. ( D ) Quantitative apoptosis ratios in PRMT6-knockdown versus control LN229 cells following 200µM TMZ treatment under normoxic or hypoxic conditions. ( E ) Rescue experiment showing HIF-1α and PRMT6 protein expression in HIF-1α-knockdown LN229 cells with PRMT6 overexpression, indicating partial restoration of HIF-1α knockdown effects. ( F ) TMZ IC50 values in the HIF-1α knockdown/PRMT6 overexpression rescue experiment. ( G ) Flow cytometry apoptosis profiles of 200µM TMZ-induced apoptosis in the HIF-1α knockdown/PRMT6 overexpression rescue model. ( H ) Quantitative apoptosis ratios in the HIF-1α knockdown/PRMT6 overexpression rescue experiment following 200µM TMZ treatment. ( I ) Rescue experiment showing PRMT6 and G3BP1 protein expression in PRMT6-knockdown LN229 cells with G3BP1 overexpression, indicating partial restoration of PRMT6 knockdown effects. ( J ) TMZ IC50 values in the PRMT6 knockdown/G3BP1 overexpression rescue experiment. ( K ) Flow cytometry apoptosis profiles of 200µM TMZ-induced apoptosis in the PRMT6 knockdown/G3BP1 overexpression rescue model. ( L ) Quantitative apoptosis ratios in the PRMT6 knockdown/G3BP1 overexpression rescue experiment following 200µM TMZ treatment. Data in B, D, F, H, J, and L are presented as mean ± SD. For A-C, E-G, I-K n = 3 biologically independent samples. Statistical significance in B, D, F, H, J, and L was assessed by one-way ANOVA followed by Tukey’s test for multiple comparisons. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: Antibodies included: Anti-PRMT6 antibody (Cat No. 14641) and anti-ADMA antibody (Cat No. 13522) (CST; Boston, Massachusetts, USA), anti-HIF-1α (Cat No. 20960-1-AP), anti-G3BP1 (Cat No. 13057-2-AP), anti-GABPA (Cat No. 21542-1-AP), and anti-BCL2L13 (Cat No. 16612-1-AP) from Proteintech (Wuhan, China); and anti-GAPDH (Cat No. bs-2188R) from Bioss (Beijing, China).

Techniques: Expressing, Knockdown, Control, Over Expression, Flow Cytometry

Journal: Journal of Translational Medicine

Article Title: Hypoxia-induced PRMT6 expression promotes temozolomide chemoresistance in glioblastoma via G3BP1

doi: 10.1186/s12967-025-07618-5

Figure Lengend Snippet: G3BP1 suppresses the translation of BCL2L13 mRNA by sequestering it into stress granules. ( A ) KEGG pathway analysis of PRMT6 expression in TCGA glioma dataset demonstrates PRMT6-mediated suppression of apoptosis pathways. ( B ) GSEA pathway analysis of PRMT6 expression in TCGA glioma dataset confirms PRMT6’s inhibitory effect on apoptotic pathways. ( C ) Heatmap analysis from CPTAC glioma proteomics database showing correlation between stress granule genes (represented by G3BP1) and apoptosis-related genes, with BCL2L13 demonstrating the most significant negative correlation with G3BP1. ( D ) BCL2L13 transcript abundance in the RNA-seq dataset ( GSE138058 ) with or without sodium arsenite stimulation. ( E ) Protein expression of G3BP1 and BCL2L13 in U251 and LN229 wild-type cells during 100µM TMZ treatment and after TMZ withdrawal. ( F ) mRNA expression of G3BP1 and BCL2L13 in U251 and LN229 wild-type cells during 100µM TMZ treatment and after TMZ withdrawal. ( G ) Protein expression of G3BP1 and BCL2L13 in G3BP1-knockdown U251 and LN229 cells following 6 h stimulation with 100µM TMZ. ( H ) mRNA expression of G3BP1 and BCL2L13 in G3BP1-knockdown U251 and LN229 cells following 6 h stimulation with 100µM TMZ. (I) RIP-qPCR analysis showing the enrichment of BCL2L13 mRNA in immunoprecipitates using an anti-G3BP1 antibody in TMZ-treated cells. (J) RNA fluorescence in situ hybridization was performed on wild-type cells using a BCL2L13 mRNA-specific probe under two conditions: untreated and following 6-hour stimulation with 100µM TMZ. Arrows indicate regions of stress granule formation and mRNA accumulation. G3BP1 is shown in green, and BCL2L13 mRNA signals are shown in red. Scale bar = 10 μm. ( K ) RNA fluorescence in situ hybridization with a BCL2L13 mRNA-specific probe was performed in both G3BP1-knockdown and corresponding control cells following TMZ treatment. Arrows indicate regions of stress granule formation and mRNA accumulation. G3BP1 is shown in green, and BCL2L13 mRNA signals are shown in red. Scale bar = 10 μm. ( L ) Schematic diagram of the proposed mechanism: hypoxia-induced PRMT6 expression promotes temozolomide chemoresistance in glioblastoma through G3BP1. Data in D-I are presented as mean ± SD. For E-K n = 3 biologically independent samples. Statistical significance in D-I was assessed by one-way ANOVA followed by Tukey’s test for multiple comparisons. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: Antibodies included: Anti-PRMT6 antibody (Cat No. 14641) and anti-ADMA antibody (Cat No. 13522) (CST; Boston, Massachusetts, USA), anti-HIF-1α (Cat No. 20960-1-AP), anti-G3BP1 (Cat No. 13057-2-AP), anti-GABPA (Cat No. 21542-1-AP), and anti-BCL2L13 (Cat No. 16612-1-AP) from Proteintech (Wuhan, China); and anti-GAPDH (Cat No. bs-2188R) from Bioss (Beijing, China).

Techniques: Expressing, RNA Sequencing, Knockdown, Fluorescence, In Situ Hybridization, Control

Journal: Journal of Translational Medicine

Article Title: Hypoxia-induced PRMT6 expression promotes temozolomide chemoresistance in glioblastoma via G3BP1

doi: 10.1186/s12967-025-07618-5

Figure Lengend Snippet: PRMT6 Promotes Glioblastoma Proliferation and Temozolomide Resistance In Vivo. ( A ) Schematic diagram of the animal experimental protocol. ( B ) The survival curve was generated from an orthotopic glioblastoma mouse model established by intracranial implantation of the following cell variants: (i) PRMT6 shCTRL + Empty Vector + DMSO; (ii) PRMT6 shRNA + Empty Vector + DMSO; (iii) PRMT6 shCTRL + Empty Vector + TMZ; (iv) PRMT6 shRNA + Empty Vector + TMZ; (v) PRMT6 shCTRL + G3BP1 OE + TMZ; and (vi) PRMT6 shRNA + G3BP1 OE + TMZ. ( C ) Representative H&E-stained brain sections showing orthotopic xenografts. Top panels: scale bar = 1.5 mm; Bottom panels: scale bar = 100 μm. ( D ) Representative immunohistochemical staining images of PRMT6, G3BP1 and BCL2L13 in intracranial tumors from nude mice. Scale bar = 100 μm. ( E ) Intracranial tumor volume was measured using brain sections from orthotopic glioblastoma mouse models. ( F ) Effects of PRMT6 and G3BP1 on TMZ-treated subcutaneous tumor growth were assessed (treatment initiation criteria: tumor volume ≈ 50 mm³). The PRMT6 shCTRL + Empty Vector + TMZ and PRMT6 shCTRL + G3BP1 OE + TMZ groups received treatment at day 7, while the PRMT6 shRNA + Empty Vector + TMZ and PRMT6 shRNA + G3BP1 OE + TMZ groups were treated at day 15. ( G ) Comparison of subcutaneous tumor sizes in nude mouse xenograft models. ( H ) Protein expression of PRMT6, G3BP1, and BCL2L13 in intracranial xenograft tissues of nude mice. Data in B, E and F are presented as mean ± SD. For B-G n = 5 biologically independent samples; H n = 3 biologically independent samples. Statistical significance in B, E and F was assessed by one-way ANOVA followed by Tukey’s test for multiple comparisons. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: Antibodies included: Anti-PRMT6 antibody (Cat No. 14641) and anti-ADMA antibody (Cat No. 13522) (CST; Boston, Massachusetts, USA), anti-HIF-1α (Cat No. 20960-1-AP), anti-G3BP1 (Cat No. 13057-2-AP), anti-GABPA (Cat No. 21542-1-AP), and anti-BCL2L13 (Cat No. 16612-1-AP) from Proteintech (Wuhan, China); and anti-GAPDH (Cat No. bs-2188R) from Bioss (Beijing, China).

Techniques: In Vivo, Generated, Plasmid Preparation, shRNA, Staining, Immunohistochemical staining, Comparison, Expressing

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: PRMT6-mediated transcriptional activation of ythdf2 promotes glioblastoma migration, invasion, and emt via the wnt–β-catenin pathway

doi: 10.1186/s13046-024-03038-3

Figure Lengend Snippet: Elevated Expression of PRMT6 in Glioma and Its Association with Poor Prognosis. A-C : Analysis of PRMT6 mRNA expression in various grades and subtypes of glioma using data from TCGA and CGGA databases. D-E : Kaplan-Meier survival analysis showing overall and progression-free survival of glioma patients with high and low PRMT6 expression in the TCGA dataset. F : Kaplan-Meier survival analysis of overall survival in glioma patients with high and low PRMT6 expression in the CGGA dataset. G-I : Analysis of PRMT6 protein and mRNA expression in normal human brain glial cell line (HEB) and glioma cell lines (LN229, U118, U87, U251). J : Clinical sample collection and analysis of PRMT6 expression in different grades of glioma patients and normal brain tissue (NBT). K-L : Representative immunohistochemistry images of PRMT6 in different grades of glioma patients and NBT. Kruskal-Wallis test and Dunn’s multiple comparisons test were used. Scale bar = 100 μm

Article Snippet: The anti-PRMT6 (Cat#14,641) antibody was from CST (Boston, MA, USA).

Techniques: Expressing, Immunohistochemistry

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: PRMT6-mediated transcriptional activation of ythdf2 promotes glioblastoma migration, invasion, and emt via the wnt–β-catenin pathway

doi: 10.1186/s13046-024-03038-3

Figure Lengend Snippet: Knockdown of PRMT6 Inhibits Migration, Invasion, and EMT in Glioma. A-B : RNA and protein expression of PRMT6 were analyzed in LN229 and U251 cell lines infected with PRMT6 shRNA or scramble shRNA lentivirus. C : Wound healing assay to evaluate the migration of LN229 and U251 cells with or without PRMT6 knockdown. D : Transwell assay assessing migration and invasion of LN229 and U251 cells with or without PRMT6 knockdown. Scale bar = 200 μm. E : Gene Set Enrichment Analysis (GSEA) based on PRMT6 expression in TCGA glioma dataset, suggesting PRMT6 promotes Epithelial-Mesenchymal Transition (EMT). F : In LN229 cells, PRMT6 knockdown leads to morphological changes resembling epithelial cell transformation, with rounder shape and fewer pseudopodia. G : Expression of EMT-related proteins E-cadherin, N-cadherin, and Vimentin in LN229 and U251 cells with or without PRMT6 knockdown

Article Snippet: The anti-PRMT6 (Cat#14,641) antibody was from CST (Boston, MA, USA).

Techniques: Migration, Expressing, Infection, shRNA, Wound Healing Assay, Transwell Assay, Transformation Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: PRMT6-mediated transcriptional activation of ythdf2 promotes glioblastoma migration, invasion, and emt via the wnt–β-catenin pathway

doi: 10.1186/s13046-024-03038-3

Figure Lengend Snippet: PRMT6 Is Associated with YTHDF2 and Promotes Its Expression. A : Differential analysis based on PRMT6 mRNA expression levels in glioma samples from the CGGA database, categorized into “high PRMT6 expression” and “low PRMT6 expression” groups. B : Correlation analysis of PRMT6 and YTHDF2 using mRNA sequencing data from glioma in the CGGA database. C : Correlation analysis of PRMT6 and YTHDF2 in glioma using mRNA sequencing data from the TCGA database. D : Correlation analysis of PRMT6 and YTHDF2 in glioma using protein data from the CPTAC database. E-F : Examination of YTHDF2 protein and mRNA expression in LN229 and U251 cells with or without PRMT6 knockdown. G-H: Analysis of YTHDF2 protein and mRNA expression in U87 and U118 cells with or without PRMT6 overexpression. I-J: Evaluation of PRMT6 protein and mRNA expression in LN229 and U251 cells with or without YTHDF2 knockdown. K-L: Assessment of PRMT6 protein and mRNA expression in LN229 and U251 cells with or without YTHDF2 overexpression

Article Snippet: The anti-PRMT6 (Cat#14,641) antibody was from CST (Boston, MA, USA).

Techniques: Expressing, Sequencing, Over Expression

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: PRMT6-mediated transcriptional activation of ythdf2 promotes glioblastoma migration, invasion, and emt via the wnt–β-catenin pathway

doi: 10.1186/s13046-024-03038-3

Figure Lengend Snippet: PRMT6 Interacts with CDK9 to Co-Regulate YTHDF2 Expression. A : Proteins specifically binding to PRMT6 in LN229 and U251 cell lines identified through mass spectrometry, intersected with transcription factors regulating YTHDF2 from the Cistrome database. B : Co-IP assay assessing the interaction between PRMT6 and CDK9 in LN229 and U251 cells. C-D : ChIP-PCR and ChIP-qPCR confirming the binding of PRMT6 and CDK9 to the YTHDF2 promoter region in LN229 cells. E : Dual-luciferase reporter assay in LN229 cells transfected with a YTHDF2 promoter-driven luciferase reporter plasmid, evaluating luciferase activity post overexpression or non-overexpression of PRMT6. F : Dual-luciferase reporter assay in HEK-293T cells, assessing luciferase activity after single or combined overexpression of PRMT6 and CDK9. G: Evaluation of CDK9 and YTHDF2 protein expression in LN229 and U251 cells with or without CDK9 knockdown. H : ChIP-qPCR analysis of CDK9 and PRMT6 binding to the YTHDF2 promoter region in LN229 cells with or without CDK9 knockdown. I: Dual-luciferase reporter assay in HEK-293T cells, measuring luciferase activity with PRMT6 knockdown, CDK9 overexpression, or CDK9 overexpression on a background of PRMT6 knockdown. J : Dual-luciferase reporter assay in HEK-293T cells, testing luciferase activity with CDK9 knockdown, PRMT6 overexpression, or PRMT6 overexpression on a background of CDK9 knockdown

Article Snippet: The anti-PRMT6 (Cat#14,641) antibody was from CST (Boston, MA, USA).

Techniques: Expressing, Binding Assay, Mass Spectrometry, Co-Immunoprecipitation Assay, Luciferase, Reporter Assay, Transfection, Plasmid Preparation, Activity Assay, Over Expression

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: PRMT6-mediated transcriptional activation of ythdf2 promotes glioblastoma migration, invasion, and emt via the wnt–β-catenin pathway

doi: 10.1186/s13046-024-03038-3

Figure Lengend Snippet: PRMT6 Facilitates Migration, Invasion, and EMT in Glioma via YTHDF2. A : Protein expression analysis of PRMT6 and YTHDF2 in LN229 and U251 cell lines, where YTHDF2 is reintroduced in a PRMT6 knockdown context. B-D : Scratch assays to assess migration rates in LN229 and U251 cells upon YTHDF2 rescue in the PRMT6 knockdown background. E-F : Transwell assays to evaluate cell migration and invasion following YTHDF2 re-expression in PRMT6 knockdown LN229 and U251 cells. Scale bar = 200 μm. G : Assessment of EMT marker proteins E-cadherin, N-cadherin, and Vimentin in LN229 and U251 cells post-rescue of YTHDF2 on a PRMT6 knockdown framework

Article Snippet: The anti-PRMT6 (Cat#14,641) antibody was from CST (Boston, MA, USA).

Techniques: Migration, Expressing, Marker

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: PRMT6-mediated transcriptional activation of ythdf2 promotes glioblastoma migration, invasion, and emt via the wnt–β-catenin pathway

doi: 10.1186/s13046-024-03038-3

Figure Lengend Snippet: PRMT6 Modulates Wnt-β-Catenin Pathway Activation via YTHDF2. A : GSEA in glioma samples from TCGA database, categorizing based on PRMT6 expression, indicates PRMT6 activation of Wnt-β-catenin pathway. B : Similar GSEA, based on YTHDF2 expression in TCGA database, suggests YTHDF2’s role in activating Wnt-β-catenin pathway. C-D : Analysis of Wnt-β-catenin pathway proteins in YTHDF2 or PRMT6 knockdown LN229 and U251 cells. F : YTHDF2 reintroduction in PRMT6 knockdown cells and its effect on Wnt-β-catenin pathway proteins. F : Illustration of m 6 A modification sites near the 3’ end of APC and GSK3β mRNA, potential YTHDF2 targets. G-H : MeRIP-qPCR analysis in LN229 cells to assess m 6 A modification levels near the 3’ end of APC and GSK3β mRNA, potentially recognized by YTHDF2, in the context of overexpression or normal expression of YTHDF2 and PRMT6, respectively. I : RIP-qPCR detection of YTHDF2 binding to APC and GSK3β mRNA in LN229 cells. J-K : mRNA stability assay measuring APC and GSK3β mRNA half-life in YTHDF2 or PRMT6 overexpressed cells

Article Snippet: The anti-PRMT6 (Cat#14,641) antibody was from CST (Boston, MA, USA).

Techniques: Activation Assay, Expressing, Modification, Over Expression, Binding Assay, Stability Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: PRMT6-mediated transcriptional activation of ythdf2 promotes glioblastoma migration, invasion, and emt via the wnt–β-catenin pathway

doi: 10.1186/s13046-024-03038-3

Figure Lengend Snippet: The Transcriptional Activation of YTHDF2 Mediated by PRMT6 Requires Its Methyltransferase Activity; Inhibiting This Activity Suppresses Glioma Cell Migration, Invasion, and EMT. A : Analysis of H3R2me2a, PRMT6, and YTHDF2 protein levels in LN229 and U251 cells treated with varying concentrations of EPZ020411 for 24 h. B : Assessment of YTHDF2 mRNA expression after 24-hour treatment with 20µM EPZ020411 in LN229 and U251 cells. C : Dual-luciferase reporter assays in LN229 or U251 cells treated with 20µM EPZ020411 for 24 h, with or without PRMT6 overexpression. D : Wound healing assay to evaluate cell migration in LN229 and U251 cells with or without EPZ020411 treatment. E-F : Transwell assays assessing cell migration and invasion with or without EPZ020411 treatment. Scale bar = 200 μm. G : Examination of EMT and Wnt-β-catenin pathway-related protein expression in LN229 and U251 cells treated with or without 20µM EPZ020411 for 24 h. H : Wild-type LN229 cells were intracranially implanted into nude mice followed by subcutaneous administration of EPZ or saline for three weeks to observe the effect of EPZ on in vivo tumorigenesis. Representative brain sections stained with H&E displayed xenograft tumors (upper panel, scale bar = 1.5 mm). In vivo invasion assays were conducted by examining the tumor margins in mouse brains (lower panel, scale bar = 100 μm). I : Tumor volumes for each group of mice were calculated. J : The relative invasive fingers of each tumor were calculated under a microscope by counting prominent and diffuse tumor tissues. K : Representative images of immunohistochemical staining for N-cadherin and E-cadherin in xenograft tumors from control and EPZ-treated groups of nude mice. Scale bar = 100 μm

Article Snippet: The anti-PRMT6 (Cat#14,641) antibody was from CST (Boston, MA, USA).

Techniques: Activation Assay, Activity Assay, Migration, Expressing, Luciferase, Over Expression, Wound Healing Assay, Saline, In Vivo, Staining, Microscopy, Immunohistochemical staining

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: PRMT6-mediated transcriptional activation of ythdf2 promotes glioblastoma migration, invasion, and emt via the wnt–β-catenin pathway

doi: 10.1186/s13046-024-03038-3

Figure Lengend Snippet: PRMT6 Promotes Glioma Invasive Growth and EMT in an Orthotopic Xenograft Model via YTHDF2. A : A nude mouse intracranial tumorigenesis assay was conducted using sh-C + Empty Vec, sh-PRMT6 + Empty Vec, sh-PRMT6 + YTHDF2, sh-C + YTHDF2, and sh-C + sh-YTHDF2 cells. Representative H&E-stained brain sections show orthotopic xenografts (top images, scale bar = 1.5 mm). Tumor margins in mouse brains were observed for in vivo invasion assessment (bottom images, scale bar = 100 μm). B : Tumor volumes were calculated for each mouse group. C : The relative invasive fingers of each tumor were calculated microscopically by counting protruding and diffused tumor tissues. D : Representative immunohistochemical staining images of PRMT6, YTHDF2, N-cadherin, and E-cadherin in mouse tumor tissues. Scale bar = 100 μm

Article Snippet: The anti-PRMT6 (Cat#14,641) antibody was from CST (Boston, MA, USA).

Techniques: Staining, In Vivo, Immunohistochemical staining

Journal: iScience

Article Title: Cooperation between PRMT1 and PRMT6 drives lung cancer health disparities among Black/African American men

doi: 10.1016/j.isci.2024.108858

Figure Lengend Snippet: Expression of PRMT1, PRMT4, and PRMT6 is elevated in NSCLC tissue, and higher PRMT6 levels are detected in LUAD tissue of Black/AA men compared to NHW men (A) Extracts of uninvolved lung tissue and tumor tissue from the same patient were analyzed by immunoblotting using anti-PRMT1, anti-PRMT4, anti-PRMT6, or anti-vinculin. A portion of Ponceau S staining for equal protein loading is shown. (B) Quantification of signals in (A) is shown. Bars indicate the mean of all samples (two-tailed t-test for p value). (C) Extracts of LUAD tissue from NHW men and Black/AA men were analyzed by immunoblotting using anti-PRMT1, anti-PRMT4, anti-PRMT6, or anti-vinculin. A portion of Ponceau S staining for equal protein loading is shown. Vinculin blots here are the same ones in Figure S6 D. (D) Quantification of signals in (C) is shown. Bars indicate the mean of all samples (two-tailed t-test for p value). (E and F) mRNA levels of PRMT1 (E) and PRMT6 (F) in LUAD tissue from NHW men and Black/AA men were analyzed by qRT-PCR. Bars indicate the mean of all samples (two-tailed t-test for p value). See also Figures S1 and .

Article Snippet: Rabbit anti-PRMT6 Antibody , Bethyl Laboratories , Cat# A300-929A; RRID: AB_2237733.

Techniques: Expressing, Western Blot, Staining, Two Tailed Test, Quantitative RT-PCR

Journal: iScience

Article Title: Cooperation between PRMT1 and PRMT6 drives lung cancer health disparities among Black/African American men

doi: 10.1016/j.isci.2024.108858

Figure Lengend Snippet: PRMT1 interacts with PRMT6 (A) H1299 cells were co-transfected with a control vector or a vector expressing FLAG-PRMT6 and vectors expressing HA-PRMT1 or HA-PRMT4. Cell extracts were subjected to anti-FLAG immunoprecipitation. The immunoprecipitates were analyzed by anti-HA or anti-FLAG immunoblotting. (B) H1299 cells were transfected with a control vector or a vector expressing FLAG-PRMT6. Cell extracts were subjected to anti-FLAG immunoprecipitation. The immunoprecipitates were analyzed by anti-PRMT1, anti-PRMT4, or anti-FLAG immunoblotting. (C) GST or GST-PRMT6 was incubated with His-PRMT1. The reactions were subjected to GST pulldown followed by anti-PRMT1 or anti-GST immunoblotting. (D) His-PRMT1 incubated with GST-PRMT6 was subjected to Ni-NTA pulldown followed by anti-PRMT6 or anti-PRMT1 immunoblotting. (E) H1299 cells were co-transfected with vectors expressing YFPn-PRMT1 and YFPc-PRMT1, YFPn-PRMT6 and YFPc-PRMT6, YFPn-PRMT6 and YFPc-PRMT1, YFPn-PRMT1 and YFPc-MS2, or YFPn-PRMT6 and YFPc-MS2. YFP signal was analyzed by fluorescence microscopy. Scale bar, 25 μm. (F) Expression of transfected proteins in (E) was analyzed by anti-FLAG or anti-HA immunoblotting. (G) Extracts of H1299 or PRMT1 KO cells were subjected to anti-PRMT6 immunoprecipitation. The immunoprecipitates were analyzed by anti-PRMT1 or anti-PRMT6 immunoblotting.

Article Snippet: Rabbit anti-PRMT6 Antibody , Bethyl Laboratories , Cat# A300-929A; RRID: AB_2237733.

Techniques: Transfection, Control, Plasmid Preparation, Expressing, Immunoprecipitation, Western Blot, Incubation, Fluorescence, Microscopy

Journal: iScience

Article Title: Cooperation between PRMT1 and PRMT6 drives lung cancer health disparities among Black/African American men

doi: 10.1016/j.isci.2024.108858

Figure Lengend Snippet: Development of a peptide inhibitor of PRMT1/PRMT6 heteromer (A) GST-PRMT1 incubated with cell extracts containing HA-PRMT1 in the presence of TAT (20 μM) or TAT-1/6i (20 μM) was subjected to GST pulldown followed by anti-HA or anti-GST immunoblotting. (B) GST-PRMT6 incubated with cell extracts containing HA-PRMT6 in the presence of TAT (20 μM) or TAT-1/6i (20 μM) was subjected to GST pulldown followed by anti-HA or anti-GST immunoblotting. (C) GST-PRMT1 incubated with cell extracts containing HA-PRMT6 in the presence of TAT (20 μM) or TAT-1/6i (20 μM) was subjected to GST pulldown followed by anti-HA or anti-GST immunoblotting. (D) GST-PRMT6 incubated with cell extracts containing HA-PRMT1 in the presence of TAT (20 μM) or TAT-1/6i (20 μM) was subjected to GST pulldown followed by anti-HA or anti-GST immunoblotting. See also Figures S3 and . (E and F) H1299 cells were co-transfected with vectors expressing YFPc-HA-PRMT1 and YFPn-FLAG-PRMT6 followed by treatment with TAT (200 μM) or TAT-1/6i (200 μM) for 24 h. Cells were subjected to anti-HA and anti-FLAG immunofluorescence. Signals were analyzed by fluorescence microscopy using 10× magnification (E, Scale bar, 100 μm) or 40× magnification (F, Scale bar, 20 μm).

Article Snippet: Rabbit anti-PRMT6 Antibody , Bethyl Laboratories , Cat# A300-929A; RRID: AB_2237733.

Techniques: Incubation, Western Blot, Transfection, Expressing, Immunofluorescence, Fluorescence, Microscopy

Journal: iScience

Article Title: Cooperation between PRMT1 and PRMT6 drives lung cancer health disparities among Black/African American men

doi: 10.1016/j.isci.2024.108858

Figure Lengend Snippet: Disruption of PRMT1/PRMT6 heteromer reduces cell growth (A and B) Extracts of H1299 (A) or H2122 (B) cells treated with TAT (20 μM), TAT-1/6i (20 μM), MS094 (an inactive control for MS023, 100 nM), or MS023 (100 nM) for 48 h were analyzed by anti-H4R3me2a, anti-H3R2me2a, anti-GAPDH, or anti-Actin immunoblotting. (C) Extracts of H1299 cells treated with TAT (20 μM) or TAT-1/6i (20 μM) for 48 h were immunoprecipitated with anti-G3BP1 or anti-PTEN. The precipitates were analyzed by anti-ADMA, anti-G3BP1, or anti-PTEN immunoblotting. (D) Extracts of A549, H1299 PRMT6 KO, H1299 PRMT1 KO, H2122, or H1299 cells were analyzed by anti-PRMT1, anti-PRMT6, or anti-GAPDH immunoblotting. (E) Reduced growth of cells expressing both PRMT1 and PRMT6 by TAT-1/6i. Cells were treated with TAT (100 μM) or TAT-1/6i (100 μM) for 2 days and cell growth was analyzed. Cells treated with TAT was set at 100%. Data are mean (n = 3) ± SD.∗∗∗∗∗, p < 0.000005 (two-tailed t-test). (F) Extracts of A549 cells transfected with a vector expressing HA-PRMT6 were analyzed by anti-HA immunoblotting. (G) Transfected A549 cells in (F) were treated with TAT (250 μM) or TAT-1/6i (250 μM) for 4 days and cell growth was analyzed. Cells treated with TAT was set at 100%. Data are mean (n = 3) ± SD. ∗, p < 0.05 (two-tailed t-test).

Article Snippet: Rabbit anti-PRMT6 Antibody , Bethyl Laboratories , Cat# A300-929A; RRID: AB_2237733.

Techniques: Disruption, Control, Western Blot, Immunoprecipitation, Expressing, Two Tailed Test, Transfection, Plasmid Preparation

Journal: iScience

Article Title: Cooperation between PRMT1 and PRMT6 drives lung cancer health disparities among Black/African American men

doi: 10.1016/j.isci.2024.108858

Figure Lengend Snippet: Disruption of PRMT1/PRMT6 heteromer inhibits the growth of LC PDXOs and PDOs (A) IHC-stained images of LC PDX tissue and PDXOs. Paraffin embedded sections of PDX and PDXOs were incubated with anti-Napsin A, anti-TTF-1, anti-CK7, or anti-p63 antibodies followed by incubation with secondary antibodies and visualized. PDX and PDXOs are positive for Napsin-A, TTF-1, and CK7 (LUAD markers) and negative for p63 (LUSC marker). Scale bar, 50 μm. (B) Extracts of H1299, H1299 1KO, H1299 6KO, PDXOs, and PDOs were analyzed by immunoblotting using anti-PRMT1, anti-PRMT6, or anti-GAPDH. (C and D) PDXOs and PDOs were treated with TAT (500 μM) or TAT-1/6i (250 or 500 μM) for 5 days. Bright-field images of treated PDXOs and PDOs were shown (C), and cell viability was analyzed (D). Scale bar, 25 μm. PDXOs and PDOs without the treatment was set at 100%. Data are mean (n = 3) ± SD. ∗, p < 0.05, ∗∗∗∗∗, p < 0.000005 (one-way ANOVA).

Article Snippet: Rabbit anti-PRMT6 Antibody , Bethyl Laboratories , Cat# A300-929A; RRID: AB_2237733.

Techniques: Disruption, Staining, Incubation, Marker, Western Blot

Journal: iScience

Article Title: Cooperation between PRMT1 and PRMT6 drives lung cancer health disparities among Black/African American men

doi: 10.1016/j.isci.2024.108858

Figure Lengend Snippet: PRMT1/PRMT6 heteromer recruits and catalyzes the arginine methylation of ILF2 (A) Methylation of ILF2 in PRMT1 KO and PRMT6 KO cells is reduced. Extracts of H1299, PRMT1 KO, or PRMT 6KO cells were immunoprecipitated with anti-ILF2. The precipitates were analyzed by anti-ADMA or anti-ILF2 immunoblotting. (B) The ability of HA-PRMT6 to catalyze methylation on ILF2 is markedly reduced in the absence of PRMT1. HA-PRMT6 expressed in H1299 or PRMT1 KO cells was immunoprecipitated with anti-HA. The precipitates were incubated with His-ILF2 in methylation assays followed by anti-ADMA or anti-HA immunoblotting. (C) The ability of HA-PRMT1 to catalyze arginine methylation on ILF2 is markedly reduced in the absence of PRMT6. HA-PRMT1 expressed in H1299 or PRMT6 KO cells was immunoprecipitated with anti-HA. The precipitates were incubated with His-ILF2 in methylation assays followed by anti-ADMA or anti-HA immunoblotting. (D) Catalytic activity of PRMT1 is required for the methylation of ILF2. FLAG-PRMT1 expressed in H1299 or PRMT1 KO cells or FLAG-PRMT1 AAA , a catalytically inactive PRMT1, expressed in PRMT1 KO cells were immunoprecipitated with anti-FLAG. The precipitates were incubated with His-ILF2 in methylation assays followed by anti-ADMA, anti-FLAG, or anti-PRMT6 immunoblotting. (E) Catalytic activity of PRMT6 is required for the methylation of ILF2. FLAG-PRMT6 expressed in H1299 or PRMT6 KO cells or FLAG-PRMT6 KLA , a catalytically inactive PRMT6, expressed in PRMT6 KO cells were immunoprecipitated with anti-FLAG. The precipitates were incubated with His-ILF2 in methylation assays followed by anti-ADMA, anti-FLAG, or anti-PRMT1 immunoblotting. (F) Disruption of PRMT1/PRMT6 heteromer decreases the methylation of ILF2. H1299, H2122 cells, or LC PDOs were treated with TAT (50 μM for H1299 and H2122, 100 μM for LC PDOs) or TAT-1/6i (50 μM for H1299 and H2122, 100 μM for LC PDOs) for 24 h (H1299 and H2122) or 5 days (LC PDOs). Extracts were immunoprecipitated with anti-ILF2 followed by anti-ADMA or anti-ILF2 immunoblotting. (G) His-ILF2 was incubated with His-PRMT1, GST-PRMT6, or both His-PRMT1 and GST-PRMT6 in methylation assays followed by anti-ADMA immunoblotting. Coomassie blue staining of the membrane after immunoblotting is shown. (H) Both His-PRMT1 and GST-PRMT6 were incubated with TAT (500 nM) or TAT-1/6i (500 nM) in room temperature for 25 min, followed by the addition of His-ILF2 and methylation assays. The reactions were analyzed by anti-ADMA immunoblotting. Coomassie blue staining of the membrane after immunoblotting is shown. (I) Coprecipitation of ILF2 with FLAG-PRMT1 is reduced in the absence of PRMT6. FLAG-PRMT1 expressed in H1299 or PRMT6 KO cells was immunoprecipitated with anti-FLAG followed by anti-ILF2 or anti-FLAG immunoblotting. (J) Coprecipitation of ILF2 with FLAG-PRMT6 is reduced in the absence of PRMT1. FLAG-PRMT6 expressed in H1299 or PRMT1 KO cells was immunoprecipitated with anti-FLAG followed by anti-ILF2 or anti-FLAG immunoblotting. (K) Coprecipitation of PRMT1 with FLAG-ILF2 is reduced in the absence of PRMT6. FLAG-ILF2 expressed in H1299 or PRMT6 KO cells was immunoprecipitated with anti-FLAG followed by anti-PRMT1 or anti-FLAG immunoblotting. (L) Coprecipitation of PRMT6 with FLAG-ILF2 is reduced in the absence of PRMT1. FLAG-ILF2 expressed in H1299 or PRMT1 KO cells was immunoprecipitated with anti-FLAG followed by anti-PRMT6 or anti-FLAG immunoblotting.

Article Snippet: Rabbit anti-PRMT6 Antibody , Bethyl Laboratories , Cat# A300-929A; RRID: AB_2237733.

Techniques: Methylation, Immunoprecipitation, Western Blot, Incubation, Activity Assay, Disruption, Staining, Membrane

Journal: iScience

Article Title: Cooperation between PRMT1 and PRMT6 drives lung cancer health disparities among Black/African American men

doi: 10.1016/j.isci.2024.108858

Figure Lengend Snippet: PRMT1/PRMT6 heteromer positively regulates ILF2 expression (A) Reduced ILF2 expression in PRMT1 KO and PRMT6 KO cells. Cell extracts were subjected to anti-ILF2 or anti-GAPDH immunoblotting. (B) Reduced ILF2 expression by TAT-1/6i. Extracts of H1299, H2122, or A549 cells treated with TAT (20 μM) or TAT-1/6i (20 μM) for 48 h were analyzed by immunoblotting using anti-ILF2, anti-GAPDH, or anti-Actin. (C) Extracts of LC PDOs treated with TAT (100 μM) or TAT-1/6i (100 μM) for 5 days were analyzed by immunoblotting using anti-ILF2 or anti-GAPDH. (D) Extracts of A549 cells transfected with a control vector or a vector expressing HA-PRMT6 were analyzed by anti-ILF2, anti-PRMT6, or anti-Actin immunoblotting. (E) A549 cells transfected with a vector expressing HA-PRMT6 were treated with control TAT (20 μM) or TAT-1/6i (20 μM) for 48 h. Cell extracts were subjected to immunoblotting with anti-ILF2, anti-PRMT6, or anti-GAPDH. (F) Decreased ILF2 protein stability in PRMT1 KO and PRMT6 KO cells. H1299, PRMT1 KO, or PRMT6 KO cells were treated with cycloheximide (50 μg/mL). Levels of ILF2 and GAPDH were analyzed 0, 2, 4, 6, and 8 h after treatment by immunoblotting. Half-life (t 1/2 ) of ILF2 was calculated and presented as mean ± SD (n = 3). (G) Decreased stability of HA-ILF2 R2/5/7/9K . HA-tagged wild-type ILF2 or ILF2 R2/5/7/9K was expressed in H1299 cells. Their stabilities were measured as in (D). Half-life of ILF2 was calculated and presented as mean ± SD (n = 3). See also Figures S5–S7 .

Article Snippet: Rabbit anti-PRMT6 Antibody , Bethyl Laboratories , Cat# A300-929A; RRID: AB_2237733.

Techniques: Expressing, Western Blot, Transfection, Control, Plasmid Preparation

Journal: iScience

Article Title: Cooperation between PRMT1 and PRMT6 drives lung cancer health disparities among Black/African American men

doi: 10.1016/j.isci.2024.108858

Figure Lengend Snippet: PRMT1/PRMT6-mediated methylation counteracts the ubiquitination of ILF2 (A) H1299, PRMT1 KO, or PRMT6 KO cells were treated with MG132 (5 μM) for 8 h. Levels of ILF2, p21, and GAPDH were analyzed by immunoblotting. (B and C) H1299 or PRMT1 KO cells (B) and H1299 or PRMT6 KO cells (C) were transfected with a vector expressing HA-ubiquitin. Cell extracts were subjected to anti-ILF2 immunoprecipitation followed by anti-HA or anti-ILF2 immunoblotting. (D) H1299 cells were co-transfected with a control vector or vectors expressing FLAG-ILF2 or FLAG-ILF2 R2/5/7/9K and a vector expressing HA-ubiquitin. Cell extracts were subjected to anti-FLAG immunoprecipitation followed by anti-HA or anti-FLAG immunoblotting. (E) H1299, PRMT1 KO, or PRMT6 KO cells were co-transfected with vectors expressing FLAG-CUL4A and HA-ILF2. Cell extracts were subjected to anti-FLAG immunoprecipitation followed by anti-FLAG or anti-HA immunoblotting. (F) H1299 cells were co-transfected with a control vector or vectors expressing FLAG-ILF2 or FLAG-ILF2 R2/5/7/9K and a vector expressing HA-CUL4A. Cell extracts were subjected to anti-HA immunoprecipitation followed by anti-HA or anti-FLAG immunoblotting. (G) Illustration of CRL4 CRBN complex and PRMT1/PRMT6 heteromer in the regulation of ILF2 expression.

Article Snippet: Rabbit anti-PRMT6 Antibody , Bethyl Laboratories , Cat# A300-929A; RRID: AB_2237733.

Techniques: Methylation, Ubiquitin Proteomics, Western Blot, Transfection, Plasmid Preparation, Expressing, Immunoprecipitation, Control

Journal: iScience

Article Title: Cooperation between PRMT1 and PRMT6 drives lung cancer health disparities among Black/African American men

doi: 10.1016/j.isci.2024.108858

Figure Lengend Snippet:

Article Snippet: Rabbit anti-PRMT6 Antibody , Bethyl Laboratories , Cat# A300-929A; RRID: AB_2237733.

Techniques: Purification, Transduction, Recombinant, Virus, Control, Polymer, SYBR Green Assay, Viability Assay, Mass Spectrometry, Sequencing, Synthesized, Plasmid Preparation, Software, Membrane, Transfection