Journal: Scientific Reports

Article Title: Loss of the major Type I arginine methyltransferase PRMT1 causes substrate scavenging by other PRMTs

doi: 10.1038/srep01311

Figure Lengend Snippet: (a) PRMT1 fl/− ER-Cre MEFs were untreated or treated with 4-hydroxytamoxifen (OHT) for 8 days. Whole cell lysates were prepared and immunoblotted with monomethylarginine (MMA1-5) and asymmetric dimethylarginine (ADMA) antibodies. MMA antibodies are highly immunoreactive towards PRMT1 knockout (−/−) MEFs compared to the wild-type (+/+) counterparts suggesting that the KO cells have a rich pool of mono-methylated proteins. ADMA antibody showed reduced immunoreactivity in PRMT1 −/− cells, which suggests that they have lower levels of dimethylated proteins. Whole cell extracts of PRMT3 (b), CARM1 (c) and PRMT6 (d) +/+ and −/− MEFs were prepared and immunoblotted with MMA1-5 antibodies. The immunoreactivity patterns are the same in +/+ versus −/− cells. PRMT3 (b), PRMT5 (e) and PRMT6 (d) MEFs were also blotted with ADMA antibody, revealing no changes in banding patterns. CARM1 (c) MEFs were immunoblotted with H3R17 antibody (H3R17me2a, Millipore). Although this antibody was originally generated to recognize dimethyl-Arg17 on histone H3, it was shown to behave as a pan-antibody . (c) It recognized a number of proteins in +/+ cells that are absent in CARM1 −/− cells, suggesting a decrease of dimethylation in these cells. (e) PRMT5 control and knockdown (KD) HeLa cells were immunoblotted with MMA1-5 and symmetric dimethylarginine (SDMA) antibodies. MMA antibodies do not show significant differences in band patterns in control versus KD cells except for a doublet of bands, which appeared at 25 kDa in KD cells (indicated with solid white arrows). SDMA antibody showed reduced immunoreactivity in KD cells. Western analyses with αPRMT1 (a), αPRMT3 (b), αCARM1 (c), αPRMT5 (e) and αPRMT6 (marked with asterisk) (d) antibodies show the loss of these PRMTs in the respective −/− cell lines. All lysates were blotted with β-actin antibody to visualize equivalent loading.

Article Snippet: The following antibodies were obtained commercially: αH3R17me2a (Millipore), CARM1 (Bethyl), PRMT6 (Bethyl) and PRMT5 (Active Motif).

Techniques: Knock-Out, Methylation, Generated, Control, Knockdown, Western Blot

Journal: Tobacco Induced Diseases

Article Title: PRMT6 mediates inflammation via activation of the NF-κB/p65 pathway on a cigarette smoke extract-induced murine emphysema model

doi: 10.18332/tid/116413

Figure Lengend Snippet: Lung function (Mean±SD)

Article Snippet: The antibodies were: PRMT6 #14641S and H3K4me3 #9727S, (Cell Signaling Technology, USA); H3R2me2a ab8046, (Abcam, USA); TNF-α Sc-8301 and IL-1β Sc-7884 (Santa Cruz Biotechnology, USA).

Techniques: Control

Journal: Tobacco Induced Diseases

Article Title: PRMT6 mediates inflammation via activation of the NF-κB/p65 pathway on a cigarette smoke extract-induced murine emphysema model

doi: 10.18332/tid/116413

Figure Lengend Snippet: Lung function and histological examination of mouse lungs. (A) Raw and Cdyn were recovered after PRMT6 treatment; n=8 mice per group. (B) HE staining of lung slides (×200) in the control group (a), CSE group (b), CSE+Lenti-NC group (c), and CSE+Lenti-PRMT6 group (d); n=4 mice per group. MLI (C) and DI (D) were calculated in each group. *p<0.05 in comparison to control group; # p<0.05 in comparison to CSE group; Δp<0.05 in comparison to CSE+Lenti-NC group

Article Snippet: The antibodies were: PRMT6 #14641S and H3K4me3 #9727S, (Cell Signaling Technology, USA); H3R2me2a ab8046, (Abcam, USA); TNF-α Sc-8301 and IL-1β Sc-7884 (Santa Cruz Biotechnology, USA).

Techniques: Staining, Control, Comparison

Journal: Tobacco Induced Diseases

Article Title: PRMT6 mediates inflammation via activation of the NF-κB/p65 pathway on a cigarette smoke extract-induced murine emphysema model

doi: 10.18332/tid/116413

Figure Lengend Snippet: Expression of epigenetic protein in mouse lung. (A) Western blot analysis showed that overexpressing of PRMT6 caused significant changes of downstream target; n=4 mice per group. C: control group, S: CSE group, N: CSE+Lenti-NC group, P: CSE+Lenti-PRMT6 group. Relative density of PRMT6 (B), H3R2me2a (C) and H3K4me3 (D) were performed. *p<0.05 in comparison to control group; # p<0.05 in comparison to CSE group; Δp<0.05 in comparison to CSE+Lenti-NC group

Article Snippet: The antibodies were: PRMT6 #14641S and H3K4me3 #9727S, (Cell Signaling Technology, USA); H3R2me2a ab8046, (Abcam, USA); TNF-α Sc-8301 and IL-1β Sc-7884 (Santa Cruz Biotechnology, USA).

Techniques: Expressing, Western Blot, Control, Comparison

Journal: Tobacco Induced Diseases

Article Title: PRMT6 mediates inflammation via activation of the NF-κB/p65 pathway on a cigarette smoke extract-induced murine emphysema model

doi: 10.18332/tid/116413

Figure Lengend Snippet: Immunohistochemical detection of NF-κB/p65 molecule and its correlation with H3K4me3. (A) Activated NF-κB/p65 were transferred into the nucleus (×400); n=4 mice per group. Dark brown nuclear cells were positive cells, shown in the control group (a), CSE group (b), CSE+Lenti-NC group (c), and CSE+Lenti-PRMT6 group (d). The percentage of positive cells (B) and its correlation with H3K4me3 (C) were performed. *p<0.05 in comparison to control group; # p<0.05 in comparison to CSE group; Δ p<0.05 in comparison to CSE+Lenti-NC group

Article Snippet: The antibodies were: PRMT6 #14641S and H3K4me3 #9727S, (Cell Signaling Technology, USA); H3R2me2a ab8046, (Abcam, USA); TNF-α Sc-8301 and IL-1β Sc-7884 (Santa Cruz Biotechnology, USA).

Techniques: Immunohistochemical staining, Control, Comparison

Journal: Tobacco Induced Diseases

Article Title: PRMT6 mediates inflammation via activation of the NF-κB/p65 pathway on a cigarette smoke extract-induced murine emphysema model

doi: 10.18332/tid/116413

Figure Lengend Snippet: Effect of PRMT6 on inflammation in mouse lung. (A) Western blot analysis showed that overexpression of PRMT6 decreased pro-inflammatory factors; n=4 mice per group. Relative density of TNF-α (B) and IL-1β (C) were performed. *p<0.05 in comparison to control group; # p<0.05 in comparison to CSE group; Δ p<0.05 in comparison to CSE+Lenti-NC group

Article Snippet: The antibodies were: PRMT6 #14641S and H3K4me3 #9727S, (Cell Signaling Technology, USA); H3R2me2a ab8046, (Abcam, USA); TNF-α Sc-8301 and IL-1β Sc-7884 (Santa Cruz Biotechnology, USA).

Techniques: Western Blot, Over Expression, Comparison, Control

Journal: Cell reports

Article Title: PRMT6 Regulates RAS/RAF Binding and MEK/ERK-Mediated Cancer Stemness Activities in Hepatocellular Carcinoma through CRAF Methylation.

doi: 10.1016/j.celrep.2018.09.053

Figure Lengend Snippet: Figure 1. PRMT6 Is Weakly Expressed and Negatively Correlated with Aggressive Cancer Features in HCC Patients (A and B) Scattered dot (A) and waterfall (B) plot analyses of PRMT6 mRNA levels in HCC and matched non-tumor liver specimens from 77 patient samples. Red and blue bars represent samples that show a relative PRMT6 fold change of R2 overexpression and underexpression, respectively (HCC/NT). (C) Scattered dot plot analysis of PRMT6 mRNA levels in normal (n = 50) and HCC (n = 371) tissues using information gathered from the TCGA HCC dataset. (D) PRMT6 immunostaining of tissue microarray comprising 83 paired non-tumor liver and HCC tissue samples. Shown are representative images of the immunostaining. Scale bar, 100 mm. (E) Graph indicates the percentage of cases displaying no and/or low or moderate and/or high staining intensity of PRMT6. (F) Proteomic expression of PRMT6 in HCC cell lines by western blot. Data are representative of two or more independent experiments. Bars and error represent mean ± SD of replicate measurements.

Article Snippet: Sections were subsequently incubated with anti-human/mouse PRMT6 (1:1000 for human, 1:100 for mouse, Abcam, ab47244), anti-human PRMT6 (1:300, Atlas Antibodies, HPA059424), anti-mouse SOX2 (1:100, R&D Systems, MAB2018) and anti-mouse p-ERK1/2 (1:100, Abcam, ab5011).

Techniques: Over Expression, Immunostaining, Microarray, Staining, Expressing, Western Blot

Journal: Cell reports

Article Title: PRMT6 Regulates RAS/RAF Binding and MEK/ERK-Mediated Cancer Stemness Activities in Hepatocellular Carcinoma through CRAF Methylation.

doi: 10.1016/j.celrep.2018.09.053

Figure Lengend Snippet: Figure 2. PRMT6 Negatively Regulates Cancer Properties in HCC (A) Representative images and quantification of number of cells that migrated and invaded in BEL7402 and Huh7 cells with or without PRMT6 expression modulated. **p < 0.01 and ***p < 0.001. Scale bar, 100 mm. (B) Percentage of annexin V-PI-positive cells in BEL7402 and Huh7 cells with or without PRMT6 expression modulated in the presence of 5-flurouracil, cisplatin, or sorafenib. (C) Xenograft tumors and tumor volume measurements of Huh7 cells with or without PRMT6 overexpressed. (D) Bioluminescence imaging and luciferase signal quantification of nude mice injected intrahepatically with luciferase-labeled BEL7402 cells with or without PRMT6 suppressed. (E) In vivo bioluminescence imaging of nude mice injected intrahepatically with luciferase-labeled MHCC97L cells with or without PRMT6 overexpressed. Ex vivo imaging of the lungs harvested. Representative H&E images of liver and lung tissues harvested. Scale bar, 100 mm. Bar chart summary of number of metastatic foci observed in lung. EV, empty vector control; NTC, non-target control; OE, overexpression. Data are representative of two or more independent experiments. Bars and error represent mean ± SD of replicate measurements.

Article Snippet: Sections were subsequently incubated with anti-human/mouse PRMT6 (1:1000 for human, 1:100 for mouse, Abcam, ab47244), anti-human PRMT6 (1:300, Atlas Antibodies, HPA059424), anti-mouse SOX2 (1:100, R&D Systems, MAB2018) and anti-mouse p-ERK1/2 (1:100, Abcam, ab5011).

Techniques: Expressing, Imaging, Luciferase, Injection, Labeling, In Vivo, Ex Vivo, Plasmid Preparation, Control, Over Expression

Journal: Cell reports

Article Title: PRMT6 Regulates RAS/RAF Binding and MEK/ERK-Mediated Cancer Stemness Activities in Hepatocellular Carcinoma through CRAF Methylation.

doi: 10.1016/j.celrep.2018.09.053

Figure Lengend Snippet: Figure 3. PRMT6 Negatively Regulates Cancer Stemness Properties in HCC (A) Oncosphere formation and serial passages of BEL7402 and Huh7 cells with or without PRMT6 expression modulated. ***p < 0.001. Scale bar, 100 mm. (B) Flow cytometry and western blot analyses for CD133 expression in Huh7 cells with or without PRMT6 overexpressed. (C) Western blot analysis for PRMT6, SOX2, and NANOG expression in BEL7402 and Huh7 cells with or without PRMT6 expression modulated. (D) Images of xenograft tumors (red arrows) formed in NOD/SCID mice injected subcutaneously with CD133+PRMT6low, CD133+PRMT6high, CD133PRMT6low, and CD133-PRMT6high cells isolated from Huh7 cells in primary passage (picture representative of 10,000 cells injected). Kaplan-Meier curves showing percentage of tumor-free survival of the annotated groups of primary and secondary recipient mice. n = 8. Engraftment rates, average tumor latency, and tumor- initiating frequency of CD133+ and CD133 subsets with or without PRMT6 overexpressed in Huh7 cells. EV, empty vector control; NTC, non-target control; OE, overexpression; TIC, tumor-initiating cells. Data are representative of two or more independent experiments. Bars and error represent mean ± SD of replicate measurements.

Article Snippet: Sections were subsequently incubated with anti-human/mouse PRMT6 (1:1000 for human, 1:100 for mouse, Abcam, ab47244), anti-human PRMT6 (1:300, Atlas Antibodies, HPA059424), anti-mouse SOX2 (1:100, R&D Systems, MAB2018) and anti-mouse p-ERK1/2 (1:100, Abcam, ab5011).

Techniques: Expressing, Flow Cytometry, Western Blot, Injection, Isolation, Plasmid Preparation, Control, Over Expression

Journal: Cell reports

Article Title: PRMT6 Regulates RAS/RAF Binding and MEK/ERK-Mediated Cancer Stemness Activities in Hepatocellular Carcinoma through CRAF Methylation.

doi: 10.1016/j.celrep.2018.09.053

Figure Lengend Snippet: Figure 4. The Catalytically Active Domain of PRMT6 Is Functionally Important in Contributing Augmented Aggressive Cancer Stemness Properties in HCC (A) Western blot analysis for ADMA expression in BEL7402 and Huh7 cells with or without PRMT6 expression modulated. (B) Schematic illustration of PRMT6 wild-type (WT) and PRMT6 catalytic inactive (mutant) constructs used in this study. (C–E) Representative images and quantification of number of cells that (C) migrated, (D) invaded, and (E) formed oncospheres in BEL7402 cells stably over- expressing EV control, WT, and mutant PRMT6. **p < 0.01 and ***p < 0.001. Scale bar, 100 mm. (F)PercentageofannexinV-PI-positivecells inBEL7402cellsstably overexpressingEVcontrol,WT,andmutantinthe presenceof5-flurouracil,cisplatin,orsorafenib. (G) Western blot analysis for PRMT6, SOX2, and NANOG expression in BEL7402 cells stably overexpressing EV control, WT, and mutant PRMT6. (H) In vivo bioluminescence imaging and luciferase signal quantification of nude mice injected intrahepatically with luciferase-labeled BEL7402 cells stably overexpressing EV control, WT, and mutant PRMT6. EV, empty vector control; mut, mutant; NTC, non-target control; OE, overexpression; WT, wild-type. Data are representative of two or more independent experiments. Bars and error represent mean ± SD of replicate measurements.

Article Snippet: Sections were subsequently incubated with anti-human/mouse PRMT6 (1:1000 for human, 1:100 for mouse, Abcam, ab47244), anti-human PRMT6 (1:300, Atlas Antibodies, HPA059424), anti-mouse SOX2 (1:100, R&D Systems, MAB2018) and anti-mouse p-ERK1/2 (1:100, Abcam, ab5011).

Techniques: Western Blot, Expressing, Mutagenesis, Construct, Stable Transfection, Control, In Vivo, Imaging, Luciferase, Injection, Labeling, Plasmid Preparation, Over Expression

Journal: Cell reports

Article Title: PRMT6 Regulates RAS/RAF Binding and MEK/ERK-Mediated Cancer Stemness Activities in Hepatocellular Carcinoma through CRAF Methylation.

doi: 10.1016/j.celrep.2018.09.053

Figure Lengend Snippet: Figure 5. PRMT6 Interacts Directly with CRAF and Methylates It on Arginine 100 Residue (A) Gene set enrichment analysis (GSEA) identified an enrichment of RAS signaling in PRMT6 silenced cells. (B) Co-immunoprecipitation analysis for validation of CRAF as a PRMT6 interacting protein partner in 293T cells expressing SFB-tagged PRMT6, 293T cells expressing SFB-tagged CRAF, BEL7402 cells expressing SFB-tagged PRMT6, and BEL7402 expressing endogenous PRMT6. (C) Left: western blot analysis for PRMT6-mediated incorporation of asymmetric arginine dimethylation in CRAF. Middle: western blot analysis of in vitro methylation assay of CRAF. Right: GAR (glycine-arginine-rich) sequence positive control in the in vitro methylation assay. (D) Schematic diagram illustrating of full-length (FL) and deletion mutations of CRAF used in this study. CRD, cysteine-rich domain; RBD, Ras binding domain. (E) Mapping of protein domain in CRAF methylated by PRMT6 through in vivo methylation assay. Red arrows indicate ADMA bands at the predicted size. White arrow indicates loss of ADMA band at the predicted size in D2 CRAF truncation mutant. Yellow arrows indicate successful FLAG pull-down. (F) Left: levels of ADMA in WT and site-directed mutants of CRAF. Right: in vitro methylation assay of CRAF R100K mutant versus WT. (G) Left: in vitro methylation assay with immunoprecipitated PRMT6 from 293T cells stably transfected with SFB-PRMT6. Assay was performed by adding no peptide, CAVFRLLHE peptide, or CAVFKLLHE mutant peptide. Right: fragmentation spectrum of the methylated peptide identified by liquid chromatography/ tandem mass spectrometry (LC-MS/MS). m/z, mass/charge ratio. ***p < 0.001. Endo, endogenous; Exo, exogenous; KD, knockdown; mut, mutant; NTC, non-target control; WT, wild-type. Data are representative of two or more independent experiments. Bars and error represent mean ± SD of replicate measurements.

Article Snippet: Sections were subsequently incubated with anti-human/mouse PRMT6 (1:1000 for human, 1:100 for mouse, Abcam, ab47244), anti-human PRMT6 (1:300, Atlas Antibodies, HPA059424), anti-mouse SOX2 (1:100, R&D Systems, MAB2018) and anti-mouse p-ERK1/2 (1:100, Abcam, ab5011).

Techniques: Residue, Immunoprecipitation, Biomarker Discovery, Expressing, Western Blot, In Vitro, Methylation, Sequencing, Positive Control, Binding Assay, In Vivo, Mutagenesis, Stable Transfection, Transfection, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Knockdown, Control

Journal: Cell reports

Article Title: PRMT6 Regulates RAS/RAF Binding and MEK/ERK-Mediated Cancer Stemness Activities in Hepatocellular Carcinoma through CRAF Methylation.

doi: 10.1016/j.celrep.2018.09.053

Figure Lengend Snippet: Figure 6. PRMT6 Methylation of CRAF at Arginine 100 Interferes with RAS/RAF Binding Domain and Inhibits MEK/ERK-Related Kinase Activity (A) CRAF kinase assay, western blot analysis for expression of phosphorylated and total MEK1/2, phosphorylated and total ERK1/2 and ERK kinase assay in BEL7402 and Huh7 cells with or without PRMT6 expression modulated. (B) Representative images and quantification of number of cells that migrated, invaded, and formed oncospheres in BEL7402 cells expressing NTC or shPRMT6 clone 956 that were treated with control or MEK inhibitor U0126. ***p < 0.001. Scale bar, 100 mm. (C) Percentage of annexin V-PI-positive cells in BEL7402 cells expressing NTC or shPRMT6 clone 956 that were treated with control or MEK inhibitor U0126, following sorafenib treatment. (D) Western blot analysis for expression of CRAF, phosphorylated and total ERK1/2, as well as ERK kinase assay in BEL7402 cells with EV control, CRAF WT, or CRAF R100K overexpressed. (E and F) Western blot analysis for the co-immunoprecipitation of CRAF and RAS in BEL7402 cells (E) with or without PRMT6 expression modulated and (F) with CRAF WT or R100K mutant. (G) In vitro methylation assay of CRAF WT and R100K mutant followed by RAS binding assay with immunoprecipitated MBP-RAS. EV, empty vector control; NTC, non-target control; OE, overexpression; WT, wild-type. Data are representative of two or more independent experiments. Bars and error represent mean ± SD of replicate measurements.

Article Snippet: Sections were subsequently incubated with anti-human/mouse PRMT6 (1:1000 for human, 1:100 for mouse, Abcam, ab47244), anti-human PRMT6 (1:300, Atlas Antibodies, HPA059424), anti-mouse SOX2 (1:100, R&D Systems, MAB2018) and anti-mouse p-ERK1/2 (1:100, Abcam, ab5011).

Techniques: Methylation, Binding Assay, Activity Assay, Kinase Assay, Western Blot, Expressing, Control, Immunoprecipitation, Mutagenesis, In Vitro, Plasmid Preparation, Over Expression

Journal: Cell reports

Article Title: PRMT6 Regulates RAS/RAF Binding and MEK/ERK-Mediated Cancer Stemness Activities in Hepatocellular Carcinoma through CRAF Methylation.

doi: 10.1016/j.celrep.2018.09.053

Figure Lengend Snippet: Figure 7. Loss of PRMT6 Expression Aggravates Liver Tumorigenesis in a DEN+CCL4 HCC-Induced PRMT6 Knockout Mouse Model, and PRMT6-Dependent CRAF/ERK Signaling Regulates HCC Stemness as Demonstrated in Patient-Derived Organoids (A) Representative pictures of livers harvested from WT and PRMT6 knockout (PRMT6/) mice that received DEN+CCl4 carcinogen induction. (B) Maximum size of HCC tumors and liver weight over body weight ratio. *p < 0.05. (C) Representative H&E and IHC images of PRMT6 and p-ERK1/2 expression of liver tissues harvested from WT or PRMT6/ mice treated with DEN+CCL4. Scale bar, 100 mm (inset, 20 mm). (D) Western blot analysis for expression of PRMT6, CD133, NANOG, SOX2, and phosphorylated and total ERK1/2 in non-tumor liver and HCC organoids with PRMT6 stably suppressed or overexpressed, respectively. (E) Percentage of viable cells in non-tumor liver organoids with PRMT6 suppressed, compared with controls, following 5-flurouracil, cisplatin, and sorafenib treatment. *p < 0.05, **p < 0.01, and ***p < 0.001. (F) Representative images and quantification of number of cells that migrated, invaded, and formed oncospheres in HCC organoids with PRMT6 overexpressed compared with controls. Scale bar, 100 mm. *p < 0.05, **p < 0.01, and ***p < 0.001. EV, empty vector control; NT, non-tumor liver; NTC, non-target control; OE, overexpression; T, tumor/HCC. Data are representative of two or more independent experiments. Bars and error represent mean ± SD of replicate measurements.

Article Snippet: Sections were subsequently incubated with anti-human/mouse PRMT6 (1:1000 for human, 1:100 for mouse, Abcam, ab47244), anti-human PRMT6 (1:300, Atlas Antibodies, HPA059424), anti-mouse SOX2 (1:100, R&D Systems, MAB2018) and anti-mouse p-ERK1/2 (1:100, Abcam, ab5011).

Techniques: Expressing, Knock-Out, Derivative Assay, Western Blot, Stable Transfection, Plasmid Preparation, Control, Over Expression

Journal: Aging (Albany NY)

Article Title: Cigarette smoke extract induces airway epithelial cell death via repressing PRMT6/AKT signaling

doi: 10.18632/aging.202210

Figure Lengend Snippet: Cigarette smoke down-regulates PRMT6 in mice lung tissues. ( A ) Balb/C mice were treated with CSE by intraperitoneal injection for three times in 1 month as described in materials and methods. Lung tissues were stained with H&E, and the emphysematous change of lung tissue was presented in CSE treated group as compared to control mice. ( B ) Mean linear intercept (MLI) and the destructive index (DI) were measured and plotted to show the histological destruction in lung tissues. ( C ) Representative staining of PRMT6 in airway epithelia and alveoli with immunohistochemistry (IHC) analysis. ( D ) Immunoblots of PRMT6 protein in mice lung tissue with GAPDH used as a loading control. Relative protein expression was measured by Image J and shown as mean ± SD. “*” denotes p < .05, based on Student t-test.

Article Snippet: PRMT6 CRISPR/Cas9 knockout (KO) plasmids and guiding RNA were designed and constructed by Santa Cruz Biotechnology Inc. BEAS-2B cells were co-transfected with PRMT6 CRISPR/Cas9 KO plasmid (Cat#: sc-403433) and HDR plasmid (Cat#: sc-403433-HDR) using UltraCruz Transfection Reagent (Cat#: sc-395739).

Techniques: Injection, Staining, Control, Immunohistochemistry, Western Blot, Expressing

Journal: Aging (Albany NY)

Article Title: Cigarette smoke extract induces airway epithelial cell death via repressing PRMT6/AKT signaling

doi: 10.18632/aging.202210

Figure Lengend Snippet: Cigarette smoke reduces the mRNA and protein expression of PRMT6 in airway epithelial cells. ( A , B ) BEAS-2B cells were treated with CSE in a range of concentrations ( A ) and time points ( B ) as indicated. Cell lysates were subjected to immunoblotting for PRMT6 and GAPDH. The densitometric results were plotted in the right panels. ( C , D ) Human primary small airway epithelial cells (HSAECs) were treated with CSE in different concentrations ( C ) for 0, 2, 4, 8h ( D ). Immunoblotting was performed to examine PRMT6 expression. Right panels showed the densitometric results of the blots. ( E ) BEAS-2B cells were exposed to cigarette smoke. Cell lysates were analyzed with PRMT6 and GAPDH via immunoblotting. RA: room air; CS: cigarette smoke. The densitometry results of the blots were plotted in the right panel. ( F ) HSAECs cells were exposed with room air (RA) or cigarette smoke (CS). Cell lysate were obtained and analyzed with PRMT6 and GAPDH immunoblotting. Right panels were the plotted densitometric results. ( G ) Total RNA was extracted from control and CSE-treated BEAS-2B cells (2%, 4%,6% for 6h, and 6% for 0, 2h, 4h, 6h). PRMT6 and GAPDH mRNA levels were determined with qRT-PCR. ( H ) HSAECs were treated with 8% CSE for 2h, 4h and 8h. Total RNA was extracted and applied to qRT-PCR to detect PRMT6 and GAPDH mRNA level. Relative PRMT6 mRNA level was plotted. Values represent mean ± SD and “*” denotes p < .05. Results were representative of at least n =3 experiments.

Article Snippet: PRMT6 CRISPR/Cas9 knockout (KO) plasmids and guiding RNA were designed and constructed by Santa Cruz Biotechnology Inc. BEAS-2B cells were co-transfected with PRMT6 CRISPR/Cas9 KO plasmid (Cat#: sc-403433) and HDR plasmid (Cat#: sc-403433-HDR) using UltraCruz Transfection Reagent (Cat#: sc-395739).

Techniques: Expressing, Western Blot, Control, Quantitative RT-PCR

Journal: Aging (Albany NY)

Article Title: Cigarette smoke extract induces airway epithelial cell death via repressing PRMT6/AKT signaling

doi: 10.18632/aging.202210

Figure Lengend Snippet: Genetic depletion of PRMT6 results in AKT dephosphorylation via impairment of PI3K-AKT signaling. ( A ) BEAS-2B cell lysates were immunoprecipitated with PRMT6 or AKT3 antibody, and the immunoprecipitates were analyzed with AKT3 and PRMT6 immunoblotting as indicated (left two panels). pcDNA3.1D-PRMT6-FLAG and pcDNA3.1D-AKT3-V5 plasmids were co-transfected into BEAS-2B cells. After 48h of transfection, cell lysates were immunoprecipitated with FLAG or V5 antibody, and the immunoprecipitants were analyzed with V5 and FLAG immunoblotting as indicated (right two panels). ( B ) PRMT6 CRISPR/Cas9 KO plasmid and HDR plasmid were applied to establish stable PRMT6 gene knockout BEAS-2B cell line. The knockout efficiency was determined by immunoblotting. The cell lysates of wild type (WT) and PRMT6 stable knockout BEAS-2B cell (PRMT6 -/- ) were applied for pAKT Thr305 , pAKT Ser472 , AKT isoforms 1, 2, 3 and PRMT6 immunoblotting (left panel). Plotted densitometry results of the pAKT Thr305 and pAKT Ser472 in WT and PRMT6 knockout group were presented (right panel). ( C, D ), Cell lysates of WT and PRMT6 -/- BEAS-2B cells were collected and immunoblotted with indicated antibodies. At the right panel of each figure, the plotted densitometry results were presented. ( E ) pcDNA3.1D-His-V5 control plasmid (Vector) and pcDNA3.1D-PRMT6-V5 plasmids were delivered into BEAS-2B cells via electroporation. After 48h, the cell lysates were collected and immunoblotted with indicated antibodies. The plotted densitometry of PI3Kp85, PDK1, PTEN and PRMT6 were presented (middle and right panel). Values represent mean ± SD and “*” denotes p < 0.05. Results were representative of at least n =3 experiments.

Article Snippet: PRMT6 CRISPR/Cas9 knockout (KO) plasmids and guiding RNA were designed and constructed by Santa Cruz Biotechnology Inc. BEAS-2B cells were co-transfected with PRMT6 CRISPR/Cas9 KO plasmid (Cat#: sc-403433) and HDR plasmid (Cat#: sc-403433-HDR) using UltraCruz Transfection Reagent (Cat#: sc-395739).

Techniques: De-Phosphorylation Assay, Immunoprecipitation, Western Blot, Transfection, CRISPR, Plasmid Preparation, Gene Knockout, Knock-Out, Control, Electroporation

Journal: Aging (Albany NY)

Article Title: Cigarette smoke extract induces airway epithelial cell death via repressing PRMT6/AKT signaling

doi: 10.18632/aging.202210

Figure Lengend Snippet: CSE downregulates PI3K/AKT signal transduction in lung epithelial cells. ( A ) BEAS-2B cells were treated with 6% CSE for 0, 2, 4, 6h. Cell lysates were immunoblotted with phosphorylated AKT and total AKT. The densitometry results of the blots were presented in the lower panel. ( B ) HSAECs cells were treated with 8% CSE in a range of concentrations as indicated for 0, 2, 4, 8h. Phosphorylated AKT and AKT3 were immunoblotted. The densitometry of the blots was presented in its lower panel. ( C ) HSAECs were applied to room air or cigarette smoke exposure. RA: room air; CS: cigarette smoke. Cell lysates were immunoblotted with pAKT Thr305 , pAKT Ser472 , AKT3 and GAPDH antibodies. The densitometry results of the blots were presented in the lower panel. ( D ) Both WT and PRMT6 -/- BEAS-2B cells were treated with CSE at different time courses. Cell lysates were subjected to immunoblotting with pAKT Thr305 , pAKT Ser472 , AKT3, PRMT6, and GAPDH. Results were shown as mean ± SD and representative of n =3 experiments. Statistical significance was indicated as *: p < .05.

Article Snippet: PRMT6 CRISPR/Cas9 knockout (KO) plasmids and guiding RNA were designed and constructed by Santa Cruz Biotechnology Inc. BEAS-2B cells were co-transfected with PRMT6 CRISPR/Cas9 KO plasmid (Cat#: sc-403433) and HDR plasmid (Cat#: sc-403433-HDR) using UltraCruz Transfection Reagent (Cat#: sc-395739).

Techniques: Transduction, Western Blot

Journal: Aging (Albany NY)

Article Title: Cigarette smoke extract induces airway epithelial cell death via repressing PRMT6/AKT signaling

doi: 10.18632/aging.202210

Figure Lengend Snippet: CSE suppresses PI3K/AKT cascade in epithelial cells. ( A , B ) BEAS-2B cells were treated with CSE at various time points as indicated. Cells lysates were analyzed with mTOR, PI3Kp110, PDK1, INPP4b, PI3Kp85, PTEN, PPP2AC, PRMT6, and GAPDH immunoblotting. The densitometry of the blots was presented in the lower panel. ( C , D ) HSAECs cells were treated with CSE at a course of time as indicated. Cells lysates were analyzed with mTOR, PI3Kp110, PDK1, INPP4b, PI3Kp85, PTEN, PPP2AC, PRMT6, and GAPDH immunoblotting. The densitometric results were plotted in the lower panels. ( E , F ) HSAECs cells were exposed to room air or cigarette smoke. Cells lysates were collected and analyzed with indicated antibodies immunoblotting. RA: room air; CS: cigarette smoke. The densitometric blots were showed in the lower panel of each figure. Results were representative of n =3 experiments. Statistical significance was indicated as *: p < .05.

Article Snippet: PRMT6 CRISPR/Cas9 knockout (KO) plasmids and guiding RNA were designed and constructed by Santa Cruz Biotechnology Inc. BEAS-2B cells were co-transfected with PRMT6 CRISPR/Cas9 KO plasmid (Cat#: sc-403433) and HDR plasmid (Cat#: sc-403433-HDR) using UltraCruz Transfection Reagent (Cat#: sc-395739).

Techniques: Western Blot

Journal: Aging (Albany NY)

Article Title: Cigarette smoke extract induces airway epithelial cell death via repressing PRMT6/AKT signaling

doi: 10.18632/aging.202210

Figure Lengend Snippet: CSE suppresses AKT phosphorylation via PRMT6/PTEN signal transduction. ( A , B ) WT and PRMT6 -/- BEAS-2B cells were treated with CSE at 2, 4, 6h. Western blotting was performed to detect the protein level of PHLPP1, PHLPP2, INPP4b, PI3Kp110, PI3Kp85, PDK1, PTEN, PRMT6 and GAPDH. ( C – E ) The plotted densitometry results of PI3Kp85, PDK1 and PTEN protein expression were presented. ( F – I ) WT and PRMT6 -/- BEAS-2B cells were treated with CSE for 2, 4, 6h. qRT-PCR was performed to detect the mRNA level of PTEN, PDK1, PIK3R1 and PIK3R2 . Results of qRT-PCR were shown as mean ± SD and representative of n =3 experiments. ( J ) Scramble siRNA and three kinds of double strands PTEN-siRNA were transfected into BEAS-2B separately. PTEN expression was detected by western blotting after transfection of PTEN-siRNA for 72hrs. Plotted PTEN protein level was presented in the right panel. ( K , L ) PTEN expression was silenced by DsiRNA in wild type and PRMT6 KO BEAS-2B cells. Western blotting was used to assay the AKT phosphorylation level. Representative blots of PTEN and PRMT6 were shown. ( M ) Cycloheximide (CHX, 100ug/ml) was applied to WT and PRMT6 -/- BEAS-2B cells for 0, 4, 8, 12h. Densitometric results of PTEN and PRMT6 blots were plotted (lower panel). Results were representative of n =3 experiments. Statistics were measured by 1-way and 2-way ANOVA, *: p < 0.05.

Article Snippet: PRMT6 CRISPR/Cas9 knockout (KO) plasmids and guiding RNA were designed and constructed by Santa Cruz Biotechnology Inc. BEAS-2B cells were co-transfected with PRMT6 CRISPR/Cas9 KO plasmid (Cat#: sc-403433) and HDR plasmid (Cat#: sc-403433-HDR) using UltraCruz Transfection Reagent (Cat#: sc-395739).

Techniques: Phospho-proteomics, Transduction, Western Blot, Expressing, Quantitative RT-PCR, Transfection

Journal: Aging (Albany NY)

Article Title: Cigarette smoke extract induces airway epithelial cell death via repressing PRMT6/AKT signaling

doi: 10.18632/aging.202210

Figure Lengend Snippet: PRMT6 overexpressed epithelia are protected from CSE driven cell death. ( A , B ) BEAS-2B cells were treated with various concentrations of CSE at indicated time points. Cell viability was measured with MTT assay. ( C ) 5×10 4 cells of WT or PRMT6 -/- cell were cultured for a variety of time points, then stained by trypan blue and counted by TC10 automatic cell counter. ( D ) The trypan blue stained cells were counted by TC10 automatic cell counter. The percentage of live cells relative to control group after CSE treatment in WT and PRMT6 -/- groups were presented. ( E ) LDH assay was used to determine cell death induced by CSE treatment in WT and PRMT6 -/- groups. Relative LDH activity indicated the epithelial cell death rate were plotted. ( F ) 3 μg of pcDNA3.1D-PRMT6-V5 plasmid and control plasmid were transfected into BEAS-2B cells via electroporation. After 48h, the vector and PRMT6 overexpressed BEAS-2B cells were treated with CSE at various time points. The percentage of live cells were counted and compared with control. ( G ) Relative LDH activity under CSE treatment in vector and PRMT6-V5 overexpressed group were determined with LDH assay. Results were shown as mean ± SD and representative of n ≥3 experiments. Statistics were measured by 1-way and 2-way ANOVA or Student t test, *p < .05 indicated the statistical significance.

Article Snippet: PRMT6 CRISPR/Cas9 knockout (KO) plasmids and guiding RNA were designed and constructed by Santa Cruz Biotechnology Inc. BEAS-2B cells were co-transfected with PRMT6 CRISPR/Cas9 KO plasmid (Cat#: sc-403433) and HDR plasmid (Cat#: sc-403433-HDR) using UltraCruz Transfection Reagent (Cat#: sc-395739).

Techniques: MTT Assay, Cell Culture, Staining, Control, Lactate Dehydrogenase Assay, Activity Assay, Plasmid Preparation, Transfection, Electroporation

Journal: Aging (Albany NY)

Article Title: Cigarette smoke extract induces airway epithelial cell death via repressing PRMT6/AKT signaling

doi: 10.18632/aging.202210

Figure Lengend Snippet: PRMT6-pAKT axis is crucial in CSE-induced epithelia death. Cigarette smoke reduces PRMT6 at both mRNA and protein levels. Decreased PRMT6 promotes PI3Kp85 expression and increases the protein stability of PTEN. Changes of PI3Kp85 and PTEN, as well as decreased PDK1, reduce the total phosphorylation level of AKT. Lower level of AKT phosphorylation in turn promotes cigarette smoke-induced lung epithelial cell death.

Article Snippet: PRMT6 CRISPR/Cas9 knockout (KO) plasmids and guiding RNA were designed and constructed by Santa Cruz Biotechnology Inc. BEAS-2B cells were co-transfected with PRMT6 CRISPR/Cas9 KO plasmid (Cat#: sc-403433) and HDR plasmid (Cat#: sc-403433-HDR) using UltraCruz Transfection Reagent (Cat#: sc-395739).

Techniques: Expressing, Phospho-proteomics

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