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: Neural Regeneration Research

Article Title: Protein arginine methyltransferase-6 regulates heterogeneous nuclear ribonucleoprotein-F expression and is a potential target for the treatment of neuropathic pain

doi: 10.4103/NRR.NRR-D-23-01539

Figure Lengend Snippet: All primers used in this study

Article Snippet: The sections were blocked overnight in 5% BSA and 1% Triton X-100 in PBS and then incubated with primary antibodies against PRMT6 (rabbit, 1:200; Novus Biologicals, Littleton, CO, USA, Cat# NB100-56642, RRID: AB_838734), heterogeneous nuclear ribonucleoprotein F (hnRNP-F) (mouse,1:200; Thermo Fisher Scientific, Waltham, MA, USA, Cat# MA5-18024, RRID: AB_2539408), calcitonin gene-related peptide (CGRP; mouse, 1:200, Abcam, Cambridge, MA, USA, Cat# ab81887, RRID: AB_1658411), isolectin B4 (IB4; 1:200, Vector Laboratories, Burlingame, CA, USA, Cat# FL-1201, RRID: AB_2314663), neurofilament-200 (NF200; mouse, 1:200, Sigma, St. Louis, MO, USA, Cat# N5389, RRID: AB_260781), glutamine synthetase (GS; mouse,1:200; Abcam, Cat# ab64613, RRID: AB_1140869), and β-tubulin III (mouse, 1:200; Abcam, Cat# ab78078, RRID: AB_2256751) for 1 hour at room temperature.

Techniques: Plasmid Preparation

Journal: Neural Regeneration Research

Article Title: Protein arginine methyltransferase-6 regulates heterogeneous nuclear ribonucleoprotein-F expression and is a potential target for the treatment of neuropathic pain

doi: 10.4103/NRR.NRR-D-23-01539

Figure Lengend Snippet: PRMT6 is mainly expressed in mouse DRG nociceptive neurons. (A) In DRG neurons, β-tubulin III (green) co-localized with PRMT6 (red). (B) Astrocyte GS (green) did not co-localize with PRMT6 (red). Nuclei were stained with DAPI (blue). (C) Distribution of PRMT6 + somata: large (18.29%), small (21.95%), and medium (60.16%). (D–F) PRMT6 + neurons were stained for NF200 (green), CGRP (green), or IB4 (green), scale bars: 50 µm. Five sections per mouse from three mice per group were evaluated. CGRP: Calcitonin gene-related peptide; DAPI: 4′,6-diamidino-2-phenylindole; DRG: dorsal root ganglion; GS: glutamine synthetase; IB4: isolectin B4; NF200: neurofilament-200; PRMT6: protein arginine methyltransferase-6.

Article Snippet: The sections were blocked overnight in 5% BSA and 1% Triton X-100 in PBS and then incubated with primary antibodies against PRMT6 (rabbit, 1:200; Novus Biologicals, Littleton, CO, USA, Cat# NB100-56642, RRID: AB_838734), heterogeneous nuclear ribonucleoprotein F (hnRNP-F) (mouse,1:200; Thermo Fisher Scientific, Waltham, MA, USA, Cat# MA5-18024, RRID: AB_2539408), calcitonin gene-related peptide (CGRP; mouse, 1:200, Abcam, Cambridge, MA, USA, Cat# ab81887, RRID: AB_1658411), isolectin B4 (IB4; 1:200, Vector Laboratories, Burlingame, CA, USA, Cat# FL-1201, RRID: AB_2314663), neurofilament-200 (NF200; mouse, 1:200, Sigma, St. Louis, MO, USA, Cat# N5389, RRID: AB_260781), glutamine synthetase (GS; mouse,1:200; Abcam, Cat# ab64613, RRID: AB_1140869), and β-tubulin III (mouse, 1:200; Abcam, Cat# ab78078, RRID: AB_2256751) for 1 hour at room temperature.

Techniques: Staining

Journal: Neural Regeneration Research

Article Title: Protein arginine methyltransferase-6 regulates heterogeneous nuclear ribonucleoprotein-F expression and is a potential target for the treatment of neuropathic pain

doi: 10.4103/NRR.NRR-D-23-01539

Figure Lengend Snippet: PRMT6 expression is reduced in the injured DRG in a mouse model of neuropathic pain. (A) Schematic diagram of the experimental procedure. (B, C) SNI increased the paw withdrawal frequency in response to stimulation with calibrated von Frey filaments (0.07 g and 0.4 g) at 3-, 7-, and 14-days post-surgery ( n = 8 mice/group). (D) Prmt6 mRNA levels decreased in the injured DRG following SNI at each time point tested ( n = 3 mice/group). (E) Western blot analysis of PRMT6 expression in the mouse ipsilateral L3/4 DRG at different time points following SNI. (F) Intensity analysis showed a marked decrease in PRMT6 expression following SNI ( n = 4 mice/group). (G, H) Representative immunofluorescence images of neurons labeled for PRMT6 in the L3/4 DRG 7 days following sham or SNI surgery. Scale bar: 50 µm. (I) Immunofluorescence analysis showed a significant decrease in the number of PRMT6-positive neurons 7 days following SNI. The data shown are from three independent experiments. ** P < 0.01, *** P < 0.001, vs . sham group (two-way analysis of variance followed by Tukey’s post hoc test for B–D, F; unpaired t -test for I). DRG: Dorsal root ganglion; H3: histone H3; PRMT6: protein arginine methyltransferase-6; qRT-PCR: quantitative reverse transcription-polymerase chain reaction; SNI: spared nerve injury.

Article Snippet: The sections were blocked overnight in 5% BSA and 1% Triton X-100 in PBS and then incubated with primary antibodies against PRMT6 (rabbit, 1:200; Novus Biologicals, Littleton, CO, USA, Cat# NB100-56642, RRID: AB_838734), heterogeneous nuclear ribonucleoprotein F (hnRNP-F) (mouse,1:200; Thermo Fisher Scientific, Waltham, MA, USA, Cat# MA5-18024, RRID: AB_2539408), calcitonin gene-related peptide (CGRP; mouse, 1:200, Abcam, Cambridge, MA, USA, Cat# ab81887, RRID: AB_1658411), isolectin B4 (IB4; 1:200, Vector Laboratories, Burlingame, CA, USA, Cat# FL-1201, RRID: AB_2314663), neurofilament-200 (NF200; mouse, 1:200, Sigma, St. Louis, MO, USA, Cat# N5389, RRID: AB_260781), glutamine synthetase (GS; mouse,1:200; Abcam, Cat# ab64613, RRID: AB_1140869), and β-tubulin III (mouse, 1:200; Abcam, Cat# ab78078, RRID: AB_2256751) for 1 hour at room temperature.

Techniques: Expressing, Western Blot, Immunofluorescence, Labeling, Quantitative RT-PCR, Reverse Transcription, Polymerase Chain Reaction

Journal: Neural Regeneration Research

Article Title: Protein arginine methyltransferase-6 regulates heterogeneous nuclear ribonucleoprotein-F expression and is a potential target for the treatment of neuropathic pain

doi: 10.4103/NRR.NRR-D-23-01539

Figure Lengend Snippet: Effects of PRMT6 overexpression on neuropathic pain induced by SNI. (A, B) Effects of LV-PRMT6 or LV-GFP microinjection into the L3/4DRG on ipsilateral and contralateral paw withdrawal frequency. (C, D) Paw withdrawal frequency following SNI at different time points ( n = 8 mice/group). (E, F) PRMT6 and H3R2me2a expression levels in mice injected with LV-PRMT6 or LV-GFP 7 days after SNI. Ipsilateral L3/4 DRG tissue was pooled from two mice as one sample ( n = 4 sample/group). (G) Images of PRMT6-positive neurons (red) in the lumbar DRG after microinjection with LV-PRMT6 or LV-GFP. Scale bar: 50 µm. (H) Immunofluorescence images showing a significant increase in the number of PRMT6-labeled neurons following injection with LV-PRMT6 ( n = 3 mice/group). (I, J) P-ERK1/2 (and ERK1/2 expression in mice injected with LV-GFP or LV-PRMT6 on day 7 following SNI. Ipsilateral L3/4 spinal cord tissue was pooled from mice ( n = 4 mice/group). The data shown are from three independent experiments. ** P < 0.01, *** P < 0.001, vs . sham + LV-GFP group; # P < 0.05, ## P < 0.01, ### P < 0.001, vs. SNI + LV-GFP group in A and B, F, H, J. Two-way analysis of variance followed by Tukey’s post hoc test was used in A–D; one-way analysis of variance followed by Tukey’s post hoc test was used in F and J; Unpaired t -test was used in H. DRG: Dorsal root ganglion; ERK1/2: extracellular signal-regulated kinase1/2; GFP: green fluorescent protein; H3: histone H3; H3R2me2a: Asymmetric dimethylation of histone H3 arginine 2; LV: lentiviral; p-ERK1/2: phospho-extracellular signal-regulated kinase 1/2; PRMT6: protein arginine methyltransferase-6; SNI: spared nerve injury.

Article Snippet: The sections were blocked overnight in 5% BSA and 1% Triton X-100 in PBS and then incubated with primary antibodies against PRMT6 (rabbit, 1:200; Novus Biologicals, Littleton, CO, USA, Cat# NB100-56642, RRID: AB_838734), heterogeneous nuclear ribonucleoprotein F (hnRNP-F) (mouse,1:200; Thermo Fisher Scientific, Waltham, MA, USA, Cat# MA5-18024, RRID: AB_2539408), calcitonin gene-related peptide (CGRP; mouse, 1:200, Abcam, Cambridge, MA, USA, Cat# ab81887, RRID: AB_1658411), isolectin B4 (IB4; 1:200, Vector Laboratories, Burlingame, CA, USA, Cat# FL-1201, RRID: AB_2314663), neurofilament-200 (NF200; mouse, 1:200, Sigma, St. Louis, MO, USA, Cat# N5389, RRID: AB_260781), glutamine synthetase (GS; mouse,1:200; Abcam, Cat# ab64613, RRID: AB_1140869), and β-tubulin III (mouse, 1:200; Abcam, Cat# ab78078, RRID: AB_2256751) for 1 hour at room temperature.

Techniques: Over Expression, Microinjection, Expressing, Injection, Immunofluorescence, Labeling

Journal: Neural Regeneration Research

Article Title: Protein arginine methyltransferase-6 regulates heterogeneous nuclear ribonucleoprotein-F expression and is a potential target for the treatment of neuropathic pain

doi: 10.4103/NRR.NRR-D-23-01539

Figure Lengend Snippet: Prmt6 –/– mice exhibit pain hypersensitivity. (A, B) Male Prmt6 –/– mice showed hyperalgesia with increased frequency of paw withdrawal in response to stimulation with calibrated von Frey filaments (0.07 and 0.4 g). (C, D) Male Prmt6 –/– mice displayed shorter paw withdrawal latencies in response to thermal stimuli than WT mice. (E, F) Female Prmt6 –/– mice showed hyperalgesia with increased frequency of paw withdrawal in response to stimulation with von Frey filaments (0.07 and 0.4 g). (G, H) Female Prmt6 –/– mice exhibited thermal allodynia compared with WT mice. (I, J) PRMT6, MOR, and H3R2me2a protein expression levels of in the dorsal root ganglion of Prmt6 –/– mice. *** P < 0.001, vs . WT group (unpaired t -test). n = 6 mice/group. The data shown are from three independent experiments. H3R2me2a: Asymmetric dimethylation of histone H3 arginine 2; MOR: μ opioid receptor; PRMT6: protein arginine methyltransferase-6; Prmt6 –/– : PRMT6 knockout; WT: wild-type.

Article Snippet: The sections were blocked overnight in 5% BSA and 1% Triton X-100 in PBS and then incubated with primary antibodies against PRMT6 (rabbit, 1:200; Novus Biologicals, Littleton, CO, USA, Cat# NB100-56642, RRID: AB_838734), heterogeneous nuclear ribonucleoprotein F (hnRNP-F) (mouse,1:200; Thermo Fisher Scientific, Waltham, MA, USA, Cat# MA5-18024, RRID: AB_2539408), calcitonin gene-related peptide (CGRP; mouse, 1:200, Abcam, Cambridge, MA, USA, Cat# ab81887, RRID: AB_1658411), isolectin B4 (IB4; 1:200, Vector Laboratories, Burlingame, CA, USA, Cat# FL-1201, RRID: AB_2314663), neurofilament-200 (NF200; mouse, 1:200, Sigma, St. Louis, MO, USA, Cat# N5389, RRID: AB_260781), glutamine synthetase (GS; mouse,1:200; Abcam, Cat# ab64613, RRID: AB_1140869), and β-tubulin III (mouse, 1:200; Abcam, Cat# ab78078, RRID: AB_2256751) for 1 hour at room temperature.

Techniques: Expressing, Knock-Out

Journal: Neural Regeneration Research

Article Title: Protein arginine methyltransferase-6 regulates heterogeneous nuclear ribonucleoprotein-F expression and is a potential target for the treatment of neuropathic pain

doi: 10.4103/NRR.NRR-D-23-01539

Figure Lengend Snippet: Prmt6 –/– mice exhibit normal innervation patterns and sensory neuron numbers. (A) L3–L4 spinal cord segments were harvested from WT or Prmt6 –/– mice and immunostained for IB4 (red) and CGRP (green) to label central nociceptive terminals. The pixel density of IB4 and CGRP in the dorsal horn of the spinal cord (expressed as arbitrary units (AUs)) was quantified in ImageJ and showed no change in central innervation density in Prmt6 –/– mice. (B) Analysis of the total number of neurons in the DRG in WT and Prmt6 –/– mice. L4–L5 DRGs were harvested from WT or Prmt6 –/– mice, and three sections from each mouse were immunostained with the pan-neural marker β-tubulin III (green) and counterstained with DAPI (blue). WT and Prmt6 –/– mice exhibited similar numbers of sensory neurons. (C) Immunostaining and quantification of WT and Prmt6 –/– mouse nerve fibers. β-tubulin III was used to label all nerve fibers, IB4 to label nonpeptidergic nociceptor fibers, and CGRP to label peptidergic nociceptor fibers. Following immunostaining, DAPI staining was performed to highlight the dermal–epidermal junction. Quantification of β-tubulin III + , IB4 + , and CGRP + nerve fibers showed that WT and Prmt6 –/– mice had similar levels of peripheral nerve density. (D) Sciatic nerves from WT and Prmt6 –/– mice were harvested and immunostained with the pan-neural marker β-tubulin III (red) and DAPI (blue). Scale bars: 200 µm in A, B, D and 100 µm in C. Quantification indicated that WT and Prmt6 –/– mice exhibited similar levels of peripheral nerve density. Unpaired t-test was used for all statistical comparisons. Five sections per mouse from four mice per group were evaluated. CGRP: Calcitonin gene-related peptide; DAPI: 4′,6-diamidino-2-phenylindole; DRG: dorsal root ganglion; IB4: isolectin B4; PRMT6: protein arginine methyltransferase-6; Prmt6 –/– : PRMT6 knockout; WT: wild-type.

Article Snippet: The sections were blocked overnight in 5% BSA and 1% Triton X-100 in PBS and then incubated with primary antibodies against PRMT6 (rabbit, 1:200; Novus Biologicals, Littleton, CO, USA, Cat# NB100-56642, RRID: AB_838734), heterogeneous nuclear ribonucleoprotein F (hnRNP-F) (mouse,1:200; Thermo Fisher Scientific, Waltham, MA, USA, Cat# MA5-18024, RRID: AB_2539408), calcitonin gene-related peptide (CGRP; mouse, 1:200, Abcam, Cambridge, MA, USA, Cat# ab81887, RRID: AB_1658411), isolectin B4 (IB4; 1:200, Vector Laboratories, Burlingame, CA, USA, Cat# FL-1201, RRID: AB_2314663), neurofilament-200 (NF200; mouse, 1:200, Sigma, St. Louis, MO, USA, Cat# N5389, RRID: AB_260781), glutamine synthetase (GS; mouse,1:200; Abcam, Cat# ab64613, RRID: AB_1140869), and β-tubulin III (mouse, 1:200; Abcam, Cat# ab78078, RRID: AB_2256751) for 1 hour at room temperature.

Techniques: Marker, Immunostaining, Staining, Knock-Out

Journal: Neural Regeneration Research

Article Title: Protein arginine methyltransferase-6 regulates heterogeneous nuclear ribonucleoprotein-F expression and is a potential target for the treatment of neuropathic pain

doi: 10.4103/NRR.NRR-D-23-01539

Figure Lengend Snippet: Effect of Prmt6 -siRNA microinjection into the DRG on nociceptive thresholds in naïve mice. (A, B) Effect of Prmt6 -siRNA or NC-siRNA microinjection into the L3/4 DRG on paw withdrawal frequency in response to mechanical stimulation ( n = 8 mice/group). (C, D) PRMT6 and H3R2me2a expression 2 days following Prmt6 -siRNA or NC-siRNA microinjection into the L3/4 DRG. Unilateral L3/4 DRG tissue was harvested from two mice as one sample for analysis ( n = 4 sample/group). (E) Representative images of PRMT6-labeled neurons in the lumbar DRG after NC-siRNA or Prmt6 -siRNA injection. Scale bar: 50 µm. (F) Immunofluorescence analysis showed a significant decrease in the number of PRMT6-positive neurons following Prmt6-siRNA injection. (G) Representative traces of the movements of mice with spontaneous continuous pain that received either saline or lidocaine during the CPP conditioning period. (H, I) Effects of unilateral microinjection of Prmt6 -siRNA or NC-siRNA into the L3/4 DRG on spontaneous continuous pain, as assessed by the CPP test. (J, K) Phospho-ERK1/2 (p-ERK1/2) and ERK1/2 expression levels in mice injected with Prmt6 -siRNA or NC-siRNA. Unilateral L3/4 spinal cord tissue was pooled from mice ( n = 3 mice/group). The data shown are from three independent experiments. ** P < 0.01, *** P < 0.001, vs. NC-ipsi group in A and B; *** P < 0.001, vs. NC group in D; ** P < 0.01, vs . sham plus LV-GFP group in F; *** P < 0.01, vs . saline-paired in H; *** P < 0.01, vs. NC in H and K. Two-way analysis of variance followed by Tukey’s post hoc test was used in A, B, and H; one-way analysis of variance followed by Tukey’s post hoc test was used in D and F; Unpaired t-test was used in H and K. con: Contralateral side; CPP: conditioned place preference; ERK1/2: extracellular signal-regulated kinase1/2; H3R2me2a: Asymmetric dimethylation of histone H3 arginine 2; ipsi: ipsilateral side; LV-GFP: lentivirus encoding green fluorescent protein; NC: negative control; p-ERK1/2: phospho-extracellular signal-regulated kinase 1/2; Prmt6 –/– : PRMT6 knockout; PRMT6: protein arginine methyltransferase-6.

Article Snippet: The sections were blocked overnight in 5% BSA and 1% Triton X-100 in PBS and then incubated with primary antibodies against PRMT6 (rabbit, 1:200; Novus Biologicals, Littleton, CO, USA, Cat# NB100-56642, RRID: AB_838734), heterogeneous nuclear ribonucleoprotein F (hnRNP-F) (mouse,1:200; Thermo Fisher Scientific, Waltham, MA, USA, Cat# MA5-18024, RRID: AB_2539408), calcitonin gene-related peptide (CGRP; mouse, 1:200, Abcam, Cambridge, MA, USA, Cat# ab81887, RRID: AB_1658411), isolectin B4 (IB4; 1:200, Vector Laboratories, Burlingame, CA, USA, Cat# FL-1201, RRID: AB_2314663), neurofilament-200 (NF200; mouse, 1:200, Sigma, St. Louis, MO, USA, Cat# N5389, RRID: AB_260781), glutamine synthetase (GS; mouse,1:200; Abcam, Cat# ab64613, RRID: AB_1140869), and β-tubulin III (mouse, 1:200; Abcam, Cat# ab78078, RRID: AB_2256751) for 1 hour at room temperature.

Techniques: Microinjection, Expressing, Labeling, Injection, Immunofluorescence, Saline, Conditioned Place Preference, Negative Control, Knock-Out

Journal: Neural Regeneration Research

Article Title: Protein arginine methyltransferase-6 regulates heterogeneous nuclear ribonucleoprotein-F expression and is a potential target for the treatment of neuropathic pain

doi: 10.4103/NRR.NRR-D-23-01539

Figure Lengend Snippet: Mean changes in locomotor function in mice

Article Snippet: The sections were blocked overnight in 5% BSA and 1% Triton X-100 in PBS and then incubated with primary antibodies against PRMT6 (rabbit, 1:200; Novus Biologicals, Littleton, CO, USA, Cat# NB100-56642, RRID: AB_838734), heterogeneous nuclear ribonucleoprotein F (hnRNP-F) (mouse,1:200; Thermo Fisher Scientific, Waltham, MA, USA, Cat# MA5-18024, RRID: AB_2539408), calcitonin gene-related peptide (CGRP; mouse, 1:200, Abcam, Cambridge, MA, USA, Cat# ab81887, RRID: AB_1658411), isolectin B4 (IB4; 1:200, Vector Laboratories, Burlingame, CA, USA, Cat# FL-1201, RRID: AB_2314663), neurofilament-200 (NF200; mouse, 1:200, Sigma, St. Louis, MO, USA, Cat# N5389, RRID: AB_260781), glutamine synthetase (GS; mouse,1:200; Abcam, Cat# ab64613, RRID: AB_1140869), and β-tubulin III (mouse, 1:200; Abcam, Cat# ab78078, RRID: AB_2256751) for 1 hour at room temperature.

Techniques:

Journal: Neural Regeneration Research

Article Title: Protein arginine methyltransferase-6 regulates heterogeneous nuclear ribonucleoprotein-F expression and is a potential target for the treatment of neuropathic pain

doi: 10.4103/NRR.NRR-D-23-01539

Figure Lengend Snippet: HnRNP-F is required for PRMT6 mediation of neuropathic pain. (A) LC-MS/MS analysis identified nine differentially expressed proteins that were common to both comparisons. (B) Heatmap showing the expression of significantly differentially expressed proteins in all samples, as determined by label-free proteomics. (C) Western blot showing that hnRNP-F expression was significantly increased at all time points following SNI ( n = 4 mice/group). (D) hnRNP-F and MOR expression in mice injected with LV-PRMT6 or LV-GFP, 7 days following SNI ( n = 4 mice/group). (E) Prmt6 -siRNA or NC-siRNA was microinjected into the L3/L4 DRG, and hnRNP-F and MOR expression were assessed 2 days later ( n = 4 mice/group). (F) Prmt6 and hnRNP-F mRNA expression following injection with LV-PRMT6 or LV-GFP, 7 days following SNI ( n = 4 mice/group). (G) Prmt6 and hnRNP-F mRNA expression in naïve mice following injection with Prmt6 -siRNA or NC-siRNA ( n = 4 mice/group). (H, I) Relative protein expression levels of PRMT6, hnRNP-F, and MOR in Neuro-2a cells treated with Prmt6 -siRNA, hnRNP-F -siRNA, or Prmt6 -siRNA and hnRNP-F -siRNA ( n = 4 repeats/group). The data shown are from three independent experiments. ** P < 0.01, *** P < 0.001, vs . sham group in C and *** P < 0.001, vs . NC group in E and G; * P < 0.05, ** P < 0.01, *** P < 0.001, vs. sham + LV-GFP group and # P < 0.05, ### P < 0.001, vs . SNI + LV-GFP group in D and F; *** P < 0. 001, vs. NC + LV-GFP group and ### P < 0.001, vs . Prmt6 -siRNA + NC group in I. Two-way analysis of variance followed by Tukey’s post hoc test was used in C; one-way analysis of variance followed by Tukey’s post hoc test was used in D, F, I; unpaired t -test was used in E, G. GFP: Green fluorescent protein; H3: histone H3; hnRNP-F: heterogeneous nuclear ribonucleoprotein F; LV: lentiviral; MOR: μ opioid receptor; NC: negative control; PRMT6: protein arginine methyltransferase-6; si-hn: hnRNP-F siRNA; si-PR: PRMT6 siRNA; SNI: spared nerve injury.

Article Snippet: The sections were blocked overnight in 5% BSA and 1% Triton X-100 in PBS and then incubated with primary antibodies against PRMT6 (rabbit, 1:200; Novus Biologicals, Littleton, CO, USA, Cat# NB100-56642, RRID: AB_838734), heterogeneous nuclear ribonucleoprotein F (hnRNP-F) (mouse,1:200; Thermo Fisher Scientific, Waltham, MA, USA, Cat# MA5-18024, RRID: AB_2539408), calcitonin gene-related peptide (CGRP; mouse, 1:200, Abcam, Cambridge, MA, USA, Cat# ab81887, RRID: AB_1658411), isolectin B4 (IB4; 1:200, Vector Laboratories, Burlingame, CA, USA, Cat# FL-1201, RRID: AB_2314663), neurofilament-200 (NF200; mouse, 1:200, Sigma, St. Louis, MO, USA, Cat# N5389, RRID: AB_260781), glutamine synthetase (GS; mouse,1:200; Abcam, Cat# ab64613, RRID: AB_1140869), and β-tubulin III (mouse, 1:200; Abcam, Cat# ab78078, RRID: AB_2256751) for 1 hour at room temperature.

Techniques: Liquid Chromatography with Mass Spectroscopy, Expressing, Western Blot, Injection, Negative Control

Journal: Neural Regeneration Research

Article Title: Protein arginine methyltransferase-6 regulates heterogeneous nuclear ribonucleoprotein-F expression and is a potential target for the treatment of neuropathic pain

doi: 10.4103/NRR.NRR-D-23-01539

Figure Lengend Snippet: hnRNP-F and PRMT6 co-localize in DRG neurons. (A) Immunofluorescence images showing co-localization of hnRNP-F (red) and PRMT6 (green) in DRG neuronal nuclei. (B) Distribution of hnRNP-F (red) within DRG neurons in Prmt6 knockout mice. Approximately 40% of β-tubulin III–positive neurons (green) were also positive for hnRNP-F immunofluorescence in WT mice. Prmt6 knockout in the DRG increased the proportion of cells exhibiting β-tubulin III and hnRNP-F colocalization to 69%. There was no significant change in the relative proportions of different neuron types. Approximately 29% of hnRNP-F-positive neurons were positive for CGRP (green), 40% for IB4 (green), and 27% for NF200 (green) in WT mice, while in Prmt6 knockout mice, approximately 30% of hnRNP-F-positive neurons were positive for CGRP, 40% for IB4, and 30% for NF200. Scale bars: 50 µm in A and B. Unpaired t -test was used. *** P < 0.001, vs . WT. Five sections per mouse from three mice per group were evaluated.

Article Snippet: The sections were blocked overnight in 5% BSA and 1% Triton X-100 in PBS and then incubated with primary antibodies against PRMT6 (rabbit, 1:200; Novus Biologicals, Littleton, CO, USA, Cat# NB100-56642, RRID: AB_838734), heterogeneous nuclear ribonucleoprotein F (hnRNP-F) (mouse,1:200; Thermo Fisher Scientific, Waltham, MA, USA, Cat# MA5-18024, RRID: AB_2539408), calcitonin gene-related peptide (CGRP; mouse, 1:200, Abcam, Cambridge, MA, USA, Cat# ab81887, RRID: AB_1658411), isolectin B4 (IB4; 1:200, Vector Laboratories, Burlingame, CA, USA, Cat# FL-1201, RRID: AB_2314663), neurofilament-200 (NF200; mouse, 1:200, Sigma, St. Louis, MO, USA, Cat# N5389, RRID: AB_260781), glutamine synthetase (GS; mouse,1:200; Abcam, Cat# ab64613, RRID: AB_1140869), and β-tubulin III (mouse, 1:200; Abcam, Cat# ab78078, RRID: AB_2256751) for 1 hour at room temperature.

Techniques: Immunofluorescence, Knock-Out

Journal: Neural Regeneration Research

Article Title: Protein arginine methyltransferase-6 regulates heterogeneous nuclear ribonucleoprotein-F expression and is a potential target for the treatment of neuropathic pain

doi: 10.4103/NRR.NRR-D-23-01539

Figure Lengend Snippet: PRMT6 regulation of hnRNP-F expression does not require methyltransferase activity but does require amino acids 319–388. (A, B) Assessment of the interaction of PRMT6 with hnRNP-F by exogenous (A) and endogenous (B) immunoprecipitation assays. (C) Western blot showing relative protein expression levels of hnRNP-F and MOR in Neuro-2a cells overexpressing PRMT6(WT) or PRMT6(dead), a catalytically inactive form of PRMT6. (D) Intensity analysis showed that PRMT6(WT) and PRMT6(dead) had similar effects on the ratio of MOR to hnRNP-F ( n = 3/group). One-way analysis of variance followed by Tukey’s post hoc test was used. *** P < 0.001, vs . hnRNP-F OE + GFP group; ### P < 0.001, vs . GFP group. (E) Structure of WT and mutant PRMT6 constructs. (F) Co-immunoprecipitation and immunoblotting analysis of HEK293T cells transfected with PRMT6-Flag and hnRNP-F-His mutants. The PRMT6-Flag mutants included PRMT6(Δ1–88), PRMT6(Δ89–188), PRMT6(Δ189–318), PRMT6(Δ319–388), and PRMT6 (dead). The data shown are from three independent experiments. GFP: Neuro-2a cells transfected with a plasmid encoding green fluorescent protein; hnRNP-F OE: Neuro-2a cells transfected with an hnRNP-F-His plasmid; hnRNP-F: heterogeneous nuclear ribonucleoprotein F; IP: immunoprecipitation; MOR: μ opioid receptor; OE: overexpression; PRMT6: protein arginine methyltransferase-6; WT: wild-type.

Article Snippet: The sections were blocked overnight in 5% BSA and 1% Triton X-100 in PBS and then incubated with primary antibodies against PRMT6 (rabbit, 1:200; Novus Biologicals, Littleton, CO, USA, Cat# NB100-56642, RRID: AB_838734), heterogeneous nuclear ribonucleoprotein F (hnRNP-F) (mouse,1:200; Thermo Fisher Scientific, Waltham, MA, USA, Cat# MA5-18024, RRID: AB_2539408), calcitonin gene-related peptide (CGRP; mouse, 1:200, Abcam, Cambridge, MA, USA, Cat# ab81887, RRID: AB_1658411), isolectin B4 (IB4; 1:200, Vector Laboratories, Burlingame, CA, USA, Cat# FL-1201, RRID: AB_2314663), neurofilament-200 (NF200; mouse, 1:200, Sigma, St. Louis, MO, USA, Cat# N5389, RRID: AB_260781), glutamine synthetase (GS; mouse,1:200; Abcam, Cat# ab64613, RRID: AB_1140869), and β-tubulin III (mouse, 1:200; Abcam, Cat# ab78078, RRID: AB_2256751) for 1 hour at room temperature.

Techniques: Expressing, Activity Assay, Immunoprecipitation, Western Blot, Mutagenesis, Construct, Transfection, Plasmid Preparation, Over Expression