Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: PRMT6 Epigenetically Drives Metabolic Switch from Fatty Acid Oxidation toward Glycolysis and Promotes Osteoclast Differentiation During Osteoporosis.

doi: 10.1002/advs.202403177

Figure Lengend Snippet: Figure 1. Omics analysis reveals an upregulation of PRMT6 in response to RANK signaling activation. A) An osteoclast differentiation model was constructed through inducing mouse bone marrow monocytes/macrophages (BMMs) with RANKL, followed by RNA-sequencing analysis. Principal Component Analysis (PCA) demonstrated distinct segregation of samples 24 h post-RANKL induction from those in pre-induction states, underscoring significant gene expression alterations driven by RANK signaling activation. B) Volcano plot illustrating differentially expressed genes (DEGs) in BMMs before and after 24 h of RANKL induction (FC ≥2, p ≤0.05). C) Gene Ontology (GO) enrichment analysis reveals significantly enriched biological pro- cesses, with a focus on GO terms related to metabolic regulation and molecular mechanisms, which are highlighted and presented. D) The Venn diagram showcasing the intersection among the ten biological processes of interest, aimed at identifying candidate genes common to both metabolic regulation and molecular mechanism control. E,F) Quantification of gene expression for Prmt6 (E) and Auts2 (F), highlighting two genes previously unidentified in osteoclastogenesis, as derived from the core of the Venn diagram analysis. G–I) In vitro gene expression analysis of Prmt6 (G), Ctsk (H), and Mmp9 (I) in RANKL-induced BMMs as assessed by quantitative PCR. J) Representative Western blot showing the expression levels of PRMT6 during the differentia- tion of BMMs into osteoclasts, induced by RANKL. K) Quantitative analysis of PRMT6 protein levels at different time points during osteoclastogenesis. L) Immunohistochemical staining showing the localization and increased expression of PRMT6 in bone sections from ovariectomized (OVX) mice com- pared to sham-operated controls. M) Quantitative analysis of PRMT6 expression levels in bone tissue, derived from immunohistochemical staining intensities.

Article Snippet: To perform IHC, the slices underwent antigen retrieval by immersion in sodium citrate buffer at 99 °C for 20 min, followed by incubation with primary antibody against Prmt6 (Santa Cruz, California, USA) overnight at 4 °C and then with HRP-conjugated secondary antibody.

Techniques: Activation Assay, Construct, RNA Sequencing, Gene Expression, Control, Derivative Assay, In Vitro, Real-time Polymerase Chain Reaction, Western Blot, Expressing, Immunohistochemical staining, Staining

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: PRMT6 Epigenetically Drives Metabolic Switch from Fatty Acid Oxidation toward Glycolysis and Promotes Osteoclast Differentiation During Osteoporosis.

doi: 10.1002/advs.202403177

Figure Lengend Snippet: Figure 2. PRMT6 deficiency inhibits RANKL-induced osteoclast differentiation and activation in vitro. A) The PRMT6 inhibitor (EPZ020411) suppressed TRAP+ multinucleated cell (TRAP+/MNC) formation and osteoclast-mediated pit formation in a dose-dependent manner. B,C) Quantitative analysis of relative area (B) or number (C) of TRAP+/MNC under the administration of 0, 5.0, and 10 μm PRMT6 inhibitor. D) Quantitative analysis of osteoclast- mediated pit formation area under the administration of PRMT6 inhibitor. E) Prmt6 knockout inhibited TRAP+ osteoclast formation and osteoclast- mediated pit formation. F,G) Quantitative analysis of relative area (F) or number (G) of TRAP+/MNC between RANKL-induced Prmt6+/+ and Prmt6−/−

Article Snippet: To perform IHC, the slices underwent antigen retrieval by immersion in sodium citrate buffer at 99 °C for 20 min, followed by incubation with primary antibody against Prmt6 (Santa Cruz, California, USA) overnight at 4 °C and then with HRP-conjugated secondary antibody.

Techniques: Activation Assay, In Vitro, Knock-Out

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: PRMT6 Epigenetically Drives Metabolic Switch from Fatty Acid Oxidation toward Glycolysis and Promotes Osteoclast Differentiation During Osteoporosis.

doi: 10.1002/advs.202403177

Figure Lengend Snippet: Figure 3. Prmt6 deficiency attenuates OVX-induced bone loss and osteoclastogenesis in vivo. A) Micro-CT images from Prmt6−/−mice and their wild- type littermates demonstrate that Prmt6 deficiency significantly mitigates OVX-induced trabecular bone loss with minimal impact on cortical bone. B) Quantitative analyses of trabecular and cortical bone parameters in femurs using micro-CT, including bone mineral density (BMD), bone volume fraction (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp), and cortical thickness (Ct. Th). C) Hematoxylin and eosin (HE) and tartrate-resistant acid phosphatase (TRAP) staining of decalcified sections of distal femurs from Prmt6−/−and their wild-type littermates. HE staining (left panels) reveals reduced OVX-induced trabecular bone loss in Prmt6−/−mice while TRAP staining (right panels) indicates fewer TRAP+ osteoclasts, marked by red arrows. D) Histomorphometric analyses of distal femurs, showing the number of osteoblasts per bone perimeter (Ob.N/B.Pm) and the osteoblast surface area per bone surface area (Ob.S/BS). E) Histomorphometric analyses of distal femurs, showing the number of osteoclasts per bone perimeter (Oc.N/B.Pm) and the osteoclast surface area per bone surface area (Oc.S/BS). F) Measurement of serum levels of C-terminal telopeptide type 1 collagen (CTX-1) in Prmt6+/+ and Prmt6−/−mice, with and without OVX treatment.

Article Snippet: To perform IHC, the slices underwent antigen retrieval by immersion in sodium citrate buffer at 99 °C for 20 min, followed by incubation with primary antibody against Prmt6 (Santa Cruz, California, USA) overnight at 4 °C and then with HRP-conjugated secondary antibody.

Techniques: In Vivo, Micro-CT, Staining

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: PRMT6 Epigenetically Drives Metabolic Switch from Fatty Acid Oxidation toward Glycolysis and Promotes Osteoclast Differentiation During Osteoporosis.

doi: 10.1002/advs.202403177

Figure Lengend Snippet: Figure 4. Prmt6 deficiency reduces the HIF-1 signaling pathway in RANKL-induced BMMs. A) ATAC-seq showed the number of accessible chromatin regions (peaks) in Prmt6−/−compared to Prmt6+/+ BMMs post-12-h RANKL stimulation, indicating altered chromatin accessibility. B) KEGG enrichment analysis for genes associated with differential peaks between Prmt6−/−and Prmt6+/+ BMMs post-12-h RANKL stimulation. A log2|Fold change| > 1 between Prmt6−/−and Prmt6+/+ RPM values indicates the differential peaks, with proximal genes considered associated. C) A Volcano plot illustrating DEGs (Fold change >= 1.2, FDR <= 0.01) between Prmt6−/−and Prmt6+/+ cells upon 24-h RANKL stimulation, derived from RNA-seq data. D) The Top 12 significantly enriched KEGG pathways based on the DEGs between Prmt6−/−and Prmt6+/+ cells, as identified from RNA-seq data, reveal key pathways impacted by Prmt6 deficiency. E) RNA-seq data analysis shows significantly downregulated pathways in the KEGG signaling transduction category and the Glycolysis/Gluconeogenesis pathway in Prmt6−/−BMMs compared to Prmt6+/+ counterparts. F) A heatmap displays genes within the HIF-1 signaling

Article Snippet: To perform IHC, the slices underwent antigen retrieval by immersion in sodium citrate buffer at 99 °C for 20 min, followed by incubation with primary antibody against Prmt6 (Santa Cruz, California, USA) overnight at 4 °C and then with HRP-conjugated secondary antibody.

Techniques: Derivative Assay, RNA Sequencing, Transduction

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: PRMT6 Epigenetically Drives Metabolic Switch from Fatty Acid Oxidation toward Glycolysis and Promotes Osteoclast Differentiation During Osteoporosis.

doi: 10.1002/advs.202403177

Figure Lengend Snippet: Figure 5. Prmt6 deficiency inhibits glycolysis in RANKL-induced BMMs. A) GSEA analysis revealed significantly inhibited glycolysis in Prmt6−/−BMMs compared to Prmt6+/+ BMMs following 24-h RANKL stimulation. B) qPCR confirmed that Prmt6 deficiency significantly reduced the expression of glycolysis-related genes in BMMs after 24 h of RANKL induction. C) Schematic of the enzymes and metabolites involved in the glycolysis pathway. D) A heatmap displayed the proteomic profiling of glycolytic enzymes, illustrating significant upregulation in response to RANKL in wild-type BMMs, an ef- fect mitigated by Prmt6 deficiency. E) Comparative analysis of glycolytic enzyme expression (HK1, PFKM/L, PKM, and LDHA) between Prmt6−/−and Prmt6+/+ BMMs during RANKL stimulation, corroborating the findings from proteomic profiling. F) Quantitative analysis shows that Prmt6 deficiency significantly inhibits RANKL-induced expression of glycolytic enzymes (HK1, PFKM/L, PKM, and LDHA). G) Quantification of lactate secretion in cell culture supernatants from Prmt6−/−and Prmt6+/+ BMMs under RANKL stimulation. H,I) Analysis of extracellular acidification rate (ECAR) revealed a decrease in Prmt6−/−BMMs compared to Prmt6+/+ counterparts after 24-h RANKL stimulation. J) Inhibition of glycolysis with 2-DG significantly abolishes the enhancement of TRAP+ osteoclast formation induced by PRMT6 overexpression during RANKL stimulation. K,L) Quantitative analysis shows that 2-DG inhibition of glycolysis significantly abolishes the relative area (K) and number (L) of TRAP+ osteoclasts induced by PRMT6 overexpres- sion. M) Western blot analysis indicates that 2-DG inhibition of glycolysis significantly abolishes the elevated protein levels of osteoclast markers ACP5, CTSK, and MMP9 induced by PRMT6 overexpression. N) Quantitative analysis demonstrates that 2-DG inhibition of glycolysis significantly abolishes the elevated protein levels of osteoclast markers ACP5, CTSK, and MMP9 induced by PRMT6 overexpression.

Article Snippet: To perform IHC, the slices underwent antigen retrieval by immersion in sodium citrate buffer at 99 °C for 20 min, followed by incubation with primary antibody against Prmt6 (Santa Cruz, California, USA) overnight at 4 °C and then with HRP-conjugated secondary antibody.

Techniques: Expressing, Cell Culture, Inhibition, Over Expression, Western Blot

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: PRMT6 Epigenetically Drives Metabolic Switch from Fatty Acid Oxidation toward Glycolysis and Promotes Osteoclast Differentiation During Osteoporosis.

doi: 10.1002/advs.202403177

Figure Lengend Snippet: Figure 6. Prmt6 deficiency increases fatty acid oxidation (FAO) in RANKL-induced BMMs. A) GSEA revealed an augmentation in FAO in Prmt6−/−

Article Snippet: To perform IHC, the slices underwent antigen retrieval by immersion in sodium citrate buffer at 99 °C for 20 min, followed by incubation with primary antibody against Prmt6 (Santa Cruz, California, USA) overnight at 4 °C and then with HRP-conjugated secondary antibody.

Techniques:

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: PRMT6 Epigenetically Drives Metabolic Switch from Fatty Acid Oxidation toward Glycolysis and Promotes Osteoclast Differentiation During Osteoporosis.

doi: 10.1002/advs.202403177

Figure Lengend Snippet: Figure 7. PRMT6 mediates metabolic shifts in RANKL-induced BMMs by regulating histone epigenetic modification. A) Heatmap from ATAC-seq analysis showing RPM across overall chromatin accessible regions (peaks) between Prmt6−/−and Prmt6+/+ BMMs after RANKL induction, suggesting increased chromatin accessibility in Prmt6−/−samples. B) A differential accessibility plot illustrating the log2 fold change in reads per accessible region against the mean reads per region, reveals that 26.3% regions exhibit increased openness (fold change > 1.3), while 11.4% show decreased accessibility (fold change < −1.3) in RANKL-stimulated Prmt6−/−BMMs compared to Prmt6+/+ counterparts. C) GSEA of RNA-seq data revealed altered histone binding in Prmt6−/−BMMs compared to Prmt6+/+ counterparts after RANKL induction, indicating the influence of PRMT6 on histone modification. D) Prmt6 deficiency counteracted RANKL-induced upregulation of H3R2me2a modification, concurrently increasing active histone mark expression (H3K27ac, H3k56ac and H3K4me3) and reducing repressive marks (H3k27me3, H3K9me3) in Prmt6−/−compared to Prmt6+/+ BMMs during RANKL stimulation. E) Quantitative analysis indicated that at 12 h post-RANKL induction, repressive histone markers were significantly lower, and active histone markers were significantly higher in Prmt6−/−BMMs compared to wild-type BMMs. F) Heatmap showing enhanced chromatin accessibility for FAO genes in Prmt6−/−compared to Prmt6+/+ BMMs after RANKL stimulation, as indicated by increased RPM of associated peaks. G) Venn diagram depicting the overlap among FAO genes demonstrating both increased chromatin accessibility (ATAC-seq) and upregulated mRNA (RNA-seq) in Prmt6−/−BMMs relative to Prmt6+/+ counterparts following RANKL induction, alongside those downregulated mRNA (RNA-seq) in Prmt6+/+ cells after RANKL treatment. H) ChIP-qPCR analysis revealing significant enrichment of H3R2me2a modification at the promoters of key FAO genes including Ppard, Cpt1a, and Acox3 in RANKL-stimulated Prmt6+/+ BMMs compared to Prmt6−/−cells. I,J) Western Blot (I) and quantitative analysis (J) show that overexpression of H3R2A,

Article Snippet: To perform IHC, the slices underwent antigen retrieval by immersion in sodium citrate buffer at 99 °C for 20 min, followed by incubation with primary antibody against Prmt6 (Santa Cruz, California, USA) overnight at 4 °C and then with HRP-conjugated secondary antibody.

Techniques: RNA Sequencing, Binding Assay, Expressing, ChIP-qPCR, Western Blot, Over Expression

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: PRMT6 Epigenetically Drives Metabolic Switch from Fatty Acid Oxidation toward Glycolysis and Promotes Osteoclast Differentiation During Osteoporosis.

doi: 10.1002/advs.202403177

Figure Lengend Snippet: Figure 8. PRMT6 inhibitor (EPZ020411) treatment protects against OVX-induced osteoporosis and osteoclastogenesis in vivo. A) Micro-CT images of femurs from Sham, OVX, and OVX mice treated with EPZ020411 at low (LD) or high dose (HD) demonstrate that EPZ treatment significantly mitigates OVX-induced trabecular bone loss, while its impact on cortical bone remains minimal. B) Micro-CT derived parameters, including bone mineral density (BMD), bone volume fraction (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp), and cortical thickness (Ct. Th) for Sham, OVX, and OVX + EPZ treated groups. C) Hematoxylin and eosin (HE) and tartrate-resistant acid phosphatase (TRAP) stained sections of distal femurs. Upper panels: HE staining reveals the attenuation of OVX-induced trabecular bone loss following EPZ treatment; Lower panels: TRAP staining, with red arrows highlighting TRAP+ osteoclasts, demonstrates a decrease in OVX-induced osteoclast formation as a result of EPZ intervention. D) Quantitative analyses of osteoblast number per bone perimeter (Ob.N/B.Pm) and osteoblast surface area per bone surface area (Ob.S/BS) in distal femurs across all groups. E) Quantitative analyses of osteoclast number per bone perimeter (Oc.N/B.Pm) and osteoclast surface area per bone surface area (Oc.S/BS) in distal femurs across all groups. F) The serum level of C-terminal telopeptide type 1 collagen (CTX-1) was measured by ELISA assay for each group.

Article Snippet: To perform IHC, the slices underwent antigen retrieval by immersion in sodium citrate buffer at 99 °C for 20 min, followed by incubation with primary antibody against Prmt6 (Santa Cruz, California, USA) overnight at 4 °C and then with HRP-conjugated secondary antibody.

Techniques: In Vivo, Micro-CT, Derivative Assay, Staining, Enzyme-linked Immunosorbent Assay

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: PRMT6 Epigenetically Drives Metabolic Switch from Fatty Acid Oxidation toward Glycolysis and Promotes Osteoclast Differentiation During Osteoporosis.

doi: 10.1002/advs.202403177

Figure Lengend Snippet: Figure 9. Schematic of PRMT6-mediated epigenetic regulation in metabolic reprogramming during osteoclastogenesis. RANKL-induced upregulation of PRMT6 leads to the asymmetric dimethylation of H3R2 at the promoters of key fatty acid oxidation (FAO) genes such as Ppard, Acox3, and Cpt1a. This epigenetic modification constricts genomic accessibility for FAO genes, thus inhibiting FAO activity. The reduction in FAO alleviates its suppressive effect on glycolysis, synergizing with PRMT6’s promotion of HIF-1 signaling-dependent glycolysis to enhance cellular glycolytic activity. Consequently, a metabolic shift from FAO to glycolysis occurs, a transition that significantly contributes to osteoclast differentiation and activation.

Article Snippet: To perform IHC, the slices underwent antigen retrieval by immersion in sodium citrate buffer at 99 °C for 20 min, followed by incubation with primary antibody against Prmt6 (Santa Cruz, California, USA) overnight at 4 °C and then with HRP-conjugated secondary antibody.

Techniques: Activity Assay, Activation Assay

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

Journal:

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Figure Lengend Snippet: PRMT6-deficient cells exhibit impaired cell movement. A, cell lysates from control and PRMT6 siRNA-treated U2OS cells were immunoblotted with α-PRMT6 and α-tubulin as a loading control. The molecular mass markers are shown on the left in kDa. B, U2OS cell migration and invasion assays were performed following PRMT6 siRNA-mediated transient knockdown. Cell migration (black bars) and cell invasion (white bars) were measured using modified Boyden chambers with filters coated with gelatin or with air-dried Matrigel, respectively. Cells that had moved to the lower surface of the filters were fixed, stained with crystal violet, and quantified as described under “Experimental Procedures.” Results are expressed as a percentage of cell movement seen in PRMT6 knockdown cells compared with control cells. Data represent the means ± S.D. of two independent experiments performed in triplicate. Statistically significant differences, as compared with respective control conditions, are indicated by **, p < 0.01 (Student's t test). WT, wild type. C, monolayer of confluent siGFP- or siPRMT6-treated U2OS cells were wounded with pipette tips, and the cells were incubated with fresh culture medium for 48 h as described under “Experimental Procedures.” The wounded areas were photographed at the beginning (0 h) and at the end (24 h) of the assay. Photographs (original magnification, ×25) obtained from a representative experiment are shown. Results are expressed as the percentage of wound healing after 24 h. Data represent the means ± S.D. of two independent experiments performed in triplicate. D, U2OS cell morphology was monitored following PRMT6 siRNA-mediated transient knockdown. siRNA-treated U2OS cells were plated on coverslips, and stress fibers and focal adhesions were visualized by phalloidin staining and α-vinculin antibodies, respectively, followed by fluorescence microscopy as described under “Experimental Procedures.” Scale bar represents 50 μm.

Article Snippet: The antibodies against PRMT6 (Western blot, A300-929A; immunoprecipitation, A300-928A) were from Bethyl Laboratories (Montgomery, TX), and antibodies against H3R2me2a (catalog number 07-585) and H3K4me3 (catalog number 9751) were from Upstate Biotechnology, Inc. (Lake Placid, NY) and Cell Signaling Technology (Pickering, Ontario, Canada), respectively.

Techniques: Migration, Modification, Staining, Transferring, Incubation, Fluorescence, Microscopy

Journal:

Article Title:

doi:

Figure Lengend Snippet: Interference with the activity of TSP-1 restores cell migration properties to PRMT6-deficient U2OS cells. A, cell lysates or the conditioned media from control and PRMT6 siRNA-treated U2OS cells were immunoblotted with α-PRMT6, α-TSP-1, and α-tubulin as a loading control. The molecular mass markers are shown on the left in kDa. B, cell migration of siGFP- and siPRMT6-treated U2OS cells was assayed in the presence of either 20 μm neutralizing TSP-1 (GGWSHW) or the control (GGYSHW) peptide in the conditioned media. The data represent the means ± S.D. of two independent experiments performed in triplicate. Statistically significant differences, as compared with respective control conditions, are indicated by **, p < 0.01 (Student's t test). C, cell migration assays were also performed with the presence of either 10 μg/ml blocking α-TSP-1 or immunoglobulin G in the conditioned media. The data represent the means ± S.D. of two independent experiments performed in triplicate. Statistically significant differences, as compared with respective control conditions, are indicated by **, p < 0.01 (Student's t test).

Article Snippet: The antibodies against PRMT6 (Western blot, A300-929A; immunoprecipitation, A300-928A) were from Bethyl Laboratories (Montgomery, TX), and antibodies against H3R2me2a (catalog number 07-585) and H3K4me3 (catalog number 9751) were from Upstate Biotechnology, Inc. (Lake Placid, NY) and Cell Signaling Technology (Pickering, Ontario, Canada), respectively.

Techniques: Activity Assay, Migration, Blocking Assay

Journal:

Article Title:

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Figure Lengend Snippet: PRMT6 regulates the activity of the TSP-1 promoter by methylating H3R2 in U2OS cells. A, activity of the TSP-1 promoter luciferase (Luc.) reporter gene was assessed in U2OS cells transfected with either siGFP or siPRMT6. The luciferase activity was normalized to Renilla, and the latter was used to assess transfection efficiency. The transfected cells were lysed and assayed for luciferase activity as described under “Experimental Procedures.” The results are expressed as a ratio of firefly luciferase (FfLuc) on Renilla luciferase (RenLuc) fold induction activities. Data represent the means ± S.D. of two independent experiments performed in triplicate. Statistically significant differences as compared with control conditions are as follows: *, p < 0.05; ***, p < 0.001 (Student's t test). B, activity of the TSP-1 promoter luciferase reporter gene was assessed in U2OS cells transfected with either empty vector (pVAX1), wild-type PRMT6 (pVAX1 PRMT6 WT), and catalytically inactive PRMT6 (pVAX1 PRMT6 VLD:KLA). The activity of a TSP-1 promoter luciferase reporter gene was assessed as in A. C, chromatin immunoprecipitation assays were performed with control IgG and α-PRMT6 antibodies. DNA oligonucleotides from the TSP-1 promoter were used in a PCR assay to amplify a DNA fragment. The latter was visualized by ethidium bromide-stained agarose gel electrophoresis. D, analysis of the occupancy of H3R2me2a and H3K4me3 histone marks at the TSP-1 gene promoter by ChIP analysis. ChIP assays were performed with chromatin prepared from U2OS cells transfected or not with control siGFP or siPRMT6. After 72 h, the cells were harvested for ChIP analysis using the indicated antibodies and a primer pair located in the promoter region of TSP-1. ChIPs experiments were performed in duplicate, and the results shown are representative of the duplicates.

Article Snippet: The antibodies against PRMT6 (Western blot, A300-929A; immunoprecipitation, A300-928A) were from Bethyl Laboratories (Montgomery, TX), and antibodies against H3R2me2a (catalog number 07-585) and H3K4me3 (catalog number 9751) were from Upstate Biotechnology, Inc. (Lake Placid, NY) and Cell Signaling Technology (Pickering, Ontario, Canada), respectively.

Techniques: Activity Assay, Luciferase, Transfection, Plasmid Preparation, Chromatin Immunoprecipitation, Staining, Agarose Gel Electrophoresis, ChIP-chip