prmt6  (Cell Signaling Technology Inc)

Journal: Advanced Science

Article Title: KDM4A Erases the H3R17me2a Mark, Facilitating Chromosome Condensation

doi: 10.1002/advs.202514281

Figure Lengend Snippet: H3R17me2a reversibly decreases to facilitate chromosome condensation during mitosis. (A), Confocal images of H3R17me2a (green) and DAPI (blue) in 10T1/2 cells at interphase and metaphase. (B), Western blots of histones from cells arrested in late G2 (RO‐3306 or BI2536), prometaphase (nocodazole), or metaphase (nocodazole release with MG132). (C), In vitro methylation assay using biotinylated histone H3 peptides (1–21 aa; AR * T * KQTARKSTGGKAPRKQLA‐GGK(biotin)) and beads‐captured PRMT. (D–F), Confocal images of H3R17me2a (green), Flag (red), and DAPI (blue) in cells overexpressing H3‐Flag‐HA‐WT or ‐R2K (D). Quantification of the chromosome width (E) and H3R17me2a intensity (F) in the metaphase plate of Flag‐positive cells. Scale bars, 5 µm. (G), Western blots of lysates from cells overexpressing H3‐Flag‐HA‐WT, ‐R2K, or ‐R2A. Red arrowheads indicate Flag‐HA‐tagged H3, whereas black arrowheads indicate endogenous H3. (H), Western blots of lysates from cells treated with nocodazole after the knockdown of CARM1 and/or PRMT6. (I), Western blots of lysates from cells treated with nocodazole for 6, 12, or 24 h after CARM1 knockdown. (J), Levels of H3S10ph measured by FACS in cells treated with CARM1 siRNA or inhibitor for 72 h. Data are presented as the mean ± SD (n = 3). (K,L), After treatment with the CARM1 inhibitor for 72 h, 10T1/2 cells stably expressing GFP‐H2B were filmed for 5 h. Representative images are shown (K), and the duration of mitosis was determined (L).

Article Snippet: The following antibodies were used for immunoblotting, immunoprecipitation, or immunostaining: actin (Santa Cruz Biotechnology, Dallas, TX, USA, sc‐47778, 1:10000), Aurora B (Abcam, Cambridge, UK, ab2254, 1:5000), Borealin (Novus Biologicals, Centennial, CO, USA, NBP1‐89951, 1:2000), CARM1 (Bethyl Laboratories, Montgomery, TX, USA, A300‐421A, 1:5000), Cyclin B1 (Cell Signaling Technology, Danvers, MA, USA, #12231, 1:2000), Flag (Cell Signaling Technology, #2368 and #8146, 1:5000), HA (Cell Signaling Technology, #3724, 1:5000), histone H3 (Cell Signaling Technology, #9715, 1:5000), H3K9me2 (EpigenTek, Farmingdale, NY, USA, #A‐4035, 1:5000), H3K9me3 (Merck Millipore, Billerica, MA, USA, 07–442, 1:5000 and Diagenode, Denville, NJ, USA, C15200146, 1:500), H3R2me2a (Merck Millipore, 07–585, 1:2000), H3R17me2a (Abcam, ab8284, 1:2000), H3R26me2a (EpigenTek, #A‐3707, 1:2000), H3S10ph (Cell Signaling Technology, #9701, 1:5000), H3T3ph (Abcam, ab78351, 1:5000), INCENP (Thermo Fisher Scientific, 39–2800, 1:2000), KDM1A (Cell Signaling Technology, #2184, 1:5000), KDM3A (Proteintech, Rosemont, IL, USA, 12835‐1‐AP, 1:2000), KDM4A (Proteintech, 29977‐1‐AP, 1:5000), KDM4C (Novus Biologicals, NBP1‐49600, 1:5000), PRMT6 (Bethyl Laboratories, Montgomery, A300‐929A, 1:2000), p‐Ser (Santa Cruz Biotechnology, sc‐81514, 1:1000), p‐Ser PKC substrate (Cell Signaling Technology, #6967, 1:2000), Sp1 (Merck Millipore, CS200631 , 1:5000), Survivin (Abcam, ab76424, 1:2000), Suv39h1 (Cell Signaling Technology, #8729, 1:2000 and Santa Cruz Biotechnology, sc‐23961, 1:500), Suv39h2 (Proteintech, 11338‐1‐AP, 1:1000), and α‐tubulin (Santa Cruz Biotechnology, sc‐5286, 1:500).

Techniques: Western Blot, In Vitro, Methylation, Knockdown, Stable Transfection, Expressing

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 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: PRMT6 and G3BP1-promoter-driven luciferase reporter plasmids and Renilla luciferase control plasmids were acquired from OBiO Technology (Shanghai, 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: PRMT6 and G3BP1-promoter-driven luciferase reporter plasmids and Renilla luciferase control plasmids were acquired from OBiO Technology (Shanghai, 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: PRMT6 and G3BP1-promoter-driven luciferase reporter plasmids and Renilla luciferase control plasmids were acquired from OBiO Technology (Shanghai, 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: PRMT6 and G3BP1-promoter-driven luciferase reporter plasmids and Renilla luciferase control plasmids were acquired from OBiO Technology (Shanghai, 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: PRMT6 and G3BP1-promoter-driven luciferase reporter plasmids and Renilla luciferase control plasmids were acquired from OBiO Technology (Shanghai, 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: PRMT6 and G3BP1-promoter-driven luciferase reporter plasmids and Renilla luciferase control plasmids were acquired from OBiO Technology (Shanghai, 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: PRMT6 and G3BP1-promoter-driven luciferase reporter plasmids and Renilla luciferase control plasmids were acquired from OBiO Technology (Shanghai, 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: PRMT6 and G3BP1-promoter-driven luciferase reporter plasmids and Renilla luciferase control plasmids were acquired from OBiO Technology (Shanghai, China).

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