prmt6  (BPS Bioscience)

Journal: Frontiers in Immunology

Article Title: PRMT6 inhibitors promote fracture healing by modulating osteoclast glucose metabolism

doi: 10.3389/fimmu.2025.1637232

Figure Lengend Snippet: PRMT6 is a key regulatory gene that can participate in the process of bone fracture healing. (A, B) Column chart and volcano plot of differentially expressed genes (DEGs) in bone callus at 1 week and 3 weeks after fracture. (C) Heatmap of gene expression values at the intersection of 1 week and 3 weeks after fracture for 20 genes. (D, E) Immunohistochemistry to verify the expression of PRMT6 in bone tissue of C57 mice as the fracture time increases. (F) Western blotting to detect changes in the expression of PRMT6 during osteoclast differentiation. (n=3, p ≤ 0.05). *p < 0.05, **p < 0.01, ***p < 0.001; "ns" is P<0.05.

Article Snippet: BMMs were treated with different concentrations of PRMT6 inhibitor (EPZ020411, MCE, USA), and when osteoclasts were fully differentiated, they were stained using a TRAP (Tartrate-Resistant Acid Phosphatase) staining kit (Servicebio) ( ).

Techniques: Gene Expression, Immunohistochemistry, Expressing, Western Blot

Journal: Frontiers in Immunology

Article Title: PRMT6 inhibitors promote fracture healing by modulating osteoclast glucose metabolism

doi: 10.3389/fimmu.2025.1637232

Figure Lengend Snippet: PRMT6 gene deficiency mice can promote fracture healing. (A) Representative photograph of PRMT6 -/- mice and PRMT6 +/+ control littermate at 8 week of age. (B) Knockout mice group and control group fracture sites were reconstructed using micro-CT on day 21. (C) Quantitative analysis of BV/TV, Tb.Th, Tb.BMD and Tb.Sp levels. (D) Representative images of HE staining. (E) Quantitative analysis of the bone trabecular area in the fracture site area. (F) Representative images of Sarfranin O-Fast Green staining. (G) Quantitative analysis of the mature bone trabecular area in the fracture site area. (male, n=5, p ≤ 0.05). *p < 0.05, **p < 0.01.

Article Snippet: BMMs were treated with different concentrations of PRMT6 inhibitor (EPZ020411, MCE, USA), and when osteoclasts were fully differentiated, they were stained using a TRAP (Tartrate-Resistant Acid Phosphatase) staining kit (Servicebio) ( ).

Techniques: Control, Knock-Out, Micro-CT, Staining

Journal: Frontiers in Immunology

Article Title: PRMT6 inhibitors promote fracture healing by modulating osteoclast glucose metabolism

doi: 10.3389/fimmu.2025.1637232

Figure Lengend Snippet: Glycolysis can play a crucial role in the process of fracture healing. (A) RNA-seq data analysis revealed the expression of KEGG signaling categories and the glycolysis pathway in Prmt6−/− fracture mice compared to Prmt6+/+ mice. (B) Based on the differentially expressed genes (DEGs) between Prmt6−/− and Prmt6+/+ fracture mice, the top 10 significantly enriched KEGG pathways were identified from the RNA-seq data. (C) GSEA analysis showed that, compared to Prmt6+/+ mice, Prmt6−/− mice exhibited significant inhibition of the glycolysis pathway three weeks after fracture. (D) WB analysis confirmed that the expression of glycolysis-related enzymes (PFKM/L, PKM, and LDHA) was inhibited in Prmt6−/− bone marrow-derived macrophages (BMMs) compared to PRMT6+/+ BMMs. (E) GSEA analysis indicated that, compared to PRMT6+/+ mice, Prmt6−/− mice exhibited significant inhibition of the NF-κB pathway three weeks after fracture. (F) WB analysis revealed that the expression of p-p65 was inhibited in Prmt6−/− bone marrow-derived macrophages (BMMs) compared to PRMT6+/+ BMMs.

Article Snippet: BMMs were treated with different concentrations of PRMT6 inhibitor (EPZ020411, MCE, USA), and when osteoclasts were fully differentiated, they were stained using a TRAP (Tartrate-Resistant Acid Phosphatase) staining kit (Servicebio) ( ).

Techniques: RNA Sequencing, Expressing, Inhibition, Derivative Assay

Journal: Frontiers in Immunology

Article Title: PRMT6 inhibitors promote fracture healing by modulating osteoclast glucose metabolism

doi: 10.3389/fimmu.2025.1637232

Figure Lengend Snippet: PRMT6 deficiency can suppress osteoclast differentiation and activation in mice BMMs. (A) Representative computer renderings of bone structure in the femurs from PRMT6 -/- and PRMT6 +/+ mice at 8 week. (male, n = 3). (B) Quantitative analysis of BV/TV, and Tb.BMD levels. (C-E) BMMs were from of PRMT6 -/- mice and PRMT6 +/+ control littermate. TRAP staining and a bone resorption assay were conducted to assess the osteoclast differentiation, quantitative analysis of the TRAP + area and bone resorption area. (F, G) Western blotting was conducted to detect the expression of MMP9, PRMT6 and CTSK. (scale bar =500μm, p ≤ 0.05). *p < 0.05, **p < 0.01.

Article Snippet: BMMs were treated with different concentrations of PRMT6 inhibitor (EPZ020411, MCE, USA), and when osteoclasts were fully differentiated, they were stained using a TRAP (Tartrate-Resistant Acid Phosphatase) staining kit (Servicebio) ( ).

Techniques: Activation Assay, Control, Staining, Western Blot, Expressing

Journal: Frontiers in Immunology

Article Title: PRMT6 inhibitors promote fracture healing by modulating osteoclast glucose metabolism

doi: 10.3389/fimmu.2025.1637232

Figure Lengend Snippet: PRMT6 inhibitor EPZ020411 can suppress osteoclast differentiation and activation in mice BMMs. (A-C) BMMs were treated with EPZ020411 of different concentrations therapy. TRAP staining and a bone resorption assay were conducted to assess the osteoclast differentiation, and quantitative analysis of the TRAP + area,number of osteoclasts and bone resorption area. (D, E) Western blotting was conducted to detect the expression of C-FOS,PRMT6 and CTSK. (F) Q-PCR analysis of gene expression levels of osteoclast markers (NFATc1 and DC-STAMP) in RANKL-induced BMMs after PRMT6 inhibition.(scale bar =200μm, p ≤ 0.05). *p < 0.05, **p < 0.01, *** p < 0.001, **** p < 0.0001; "ns" is P<0.05.

Article Snippet: BMMs were treated with different concentrations of PRMT6 inhibitor (EPZ020411, MCE, USA), and when osteoclasts were fully differentiated, they were stained using a TRAP (Tartrate-Resistant Acid Phosphatase) staining kit (Servicebio) ( ).

Techniques: Activation Assay, Staining, Western Blot, Expressing, Gene Expression, Inhibition

Journal: Frontiers in Immunology

Article Title: PRMT6 inhibitors promote fracture healing by modulating osteoclast glucose metabolism

doi: 10.3389/fimmu.2025.1637232

Figure Lengend Snippet: PRMT6 inhibitor EPZ020411 can accelerate the process of fracture healing (A) Using micro-CT reconstruction images of the fracture site at 21 days after fracture in mice injected with PBS and mice injected with the inhibitor EPZ020411. (male, n = 3). (B, C) Quantitative analysis of BV/TV, Tb.Th, Tb.BMD and Tb.Sp levels. (D, E) Representative HE staining images of the inhibitor group and the PBS group, quantitative analysis of the bone trabecular area. (F, G) Representative Sarfranin O-Fast Green staining images of the inhibitor group and the PBS group, quantitative analysis of the chondrocyte area.

Article Snippet: BMMs were treated with different concentrations of PRMT6 inhibitor (EPZ020411, MCE, USA), and when osteoclasts were fully differentiated, they were stained using a TRAP (Tartrate-Resistant Acid Phosphatase) staining kit (Servicebio) ( ).

Techniques: Micro-CT, Injection, 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: 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