Journal: Communications Biology

Article Title: MEP50/PRMT5-mediated methylation activates GLI1 in Hedgehog signalling through inhibition of ubiquitination by the ITCH/NUMB complex

doi: 10.1038/s42003-018-0275-4

Figure Lengend Snippet: GLI1 interacts with the MEP50/PRMT5 complex. a FLAG-GLI1 interacted with endogenous MEP50 and interaction of FLAG-GLI1 and MEP50 was increased by HH signalling pathway activation. C3H10T1/2 cells were transfected with FLAG-GLI1 or the empty vector for 24 h and then treated with 300 nM SAG for an additional 24 h. Interaction of FLAG-GLI1 and MEP50 was detected by immunoprecipitation with anti-FLAG antibody followed by immunoblot analysis using anti-FLAG and anti-MEP50 antibodies. b Schematic structures of MEP50 deletion mutants. c Mapping of the GLI1-binding region in MEP50 by immunoprecipitation analysis. HEK293T cells were transfected with Myc-MEP50 deletion mutants and FLAG-GLI1 plasmids for 24 h. Interaction of FLAG-GLI1 and Myc-MEP50 deletion mutants was detected by immunoprecipitation with anti-FLAG antibody followed by immunoblot analysis using anti-FLAG and anti-Myc antibodies. d Schematic of GLI1 deletion mutants. e GST pull-down assays to map the MEP50-binding region in GLI1. GST-GLI1 deletion mutants coupled to glutathione sepharose were incubated with immunoprecipitated Myc-MEP50 from HEK293T cells. Immunoblotting was performed with an anti-Myc antibody. In a and e , data represent one of three independent experiments with similar results. In c , data represent one of two independent experiments with similar results. Unprocessed original scans of blots are shown in Supplementary Fig.

Article Snippet: The TaqMan gene expression assays used for mouse samples were Ptch1 (Mm01306905_m1), Gli1 (Mm00494645_m1), Foxm1 (Mm00514924_m1), Bcl2 (Mm00477631_m1), Mep50 (Mm01296589_g1), and Prmt5 (Mm00550472_m1).

Techniques: Activation Assay, Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot, Binding Assay, Incubation

Journal: Communications Biology

Article Title: MEP50/PRMT5-mediated methylation activates GLI1 in Hedgehog signalling through inhibition of ubiquitination by the ITCH/NUMB complex

doi: 10.1038/s42003-018-0275-4

Figure Lengend Snippet: MEP50/PRMT5 complex supports GLI1 activation through GLI1 stabilisation downstream of the HH signalling pathway. a–c Endogenous GLI1/MEP50/PRMT5 complex in C3H10T1/2 cells. Cells were treated with SAG for 36 h, and complex was detected by immunoprecipitation (IP) with anti-PRMT5 (D5P2T) ( a ), anti-MEP50 (ERP10708 [B]) ( b ), or anti-GLI1 (V812) ( c ) antibodies, followed by immunoblot (IB) with antibodies against indicated proteins. d Dissociation of PRMT5 and GLI1 in stable MEP50 knockdown C3H10T1/2 cells by siMEP50-m2. Cells were treated with SAG for 48 h and treated with 50 μM MG132 for 4 h. GLI1/PRMT5 complex was detected by immunoprecipitation with anti-PRMT5 (D5P2T) or anti-GLI1 (V812) antibodies, followed by immunoblot with indicated antibodies. e Immunoblot of endogenous GLI1 in C3H10T1/2 cells expressing MEP50 siRNAs (siMEP50-m1 or siMEP50-m2). f Immunoblot of endogenous GLI1 in C3H10T1/2 cells expressing two independent PRMT5 siRNAs. g Immunoblot of nuclear and cytoplasmic GLI1 and MEP50 in stable MEP50-knockdown (siMEP50-m2) or control siGFP-expressing cells treated with 300 nM SAG. Cells were treated with SAG for 24 h and separated into cytosol and nucleus fractions. h Immunoblot analysis of endogenous nuclear and cytoplasmic GLI1 and PRMT5 in stable PRMT5-knockdown (siPRMT5-m2) C3H10T1/2 cells. Cells were treated with 300 nM SAG for 24 h and then separated as in h . i In vivo ubiquitination of GLI1 in C3H10T1/2 cells with or without expression of siMEP50. FLAG-ubiquitin was transfected into C3H10T1/2 cells. After 48 h of transfection, then cells were treated with 50 µM MG132 for 4 h. Endogenous ubiquitinated GLI1 was immunoprecipitated with an anti-GLI1 (C-1) antibody, followed by immunoblotting with indicated antibodies. j In vivo ubiquitination of GLI1 in C3H10T1/2 cells with exogenous expression of PRMT5 or MEP50. FLAG-ubiquitin and HA-PRMT5, HA-PRMT5 G367A/R368A (inactive form of PRMT5), or Myc-MEP50 were transfected into C3H10T1/2 cells. After 48 h, the cells were treated with 50 µM MG132 for 4 h. Endogenous ubiquitinated GLI1 was detected as described in i . In a , i and j , data represent one of two independent experiments with similar results. Unprocessed original scans of blots are shown in Supplementary Fig.

Article Snippet: The TaqMan gene expression assays used for mouse samples were Ptch1 (Mm01306905_m1), Gli1 (Mm00494645_m1), Foxm1 (Mm00514924_m1), Bcl2 (Mm00477631_m1), Mep50 (Mm01296589_g1), and Prmt5 (Mm00550472_m1).

Techniques: Activation Assay, Immunoprecipitation, Western Blot, Knockdown, Expressing, Control, In Vivo, Ubiquitin Proteomics, Transfection

Journal: Communications Biology

Article Title: MEP50/PRMT5-mediated methylation activates GLI1 in Hedgehog signalling through inhibition of ubiquitination by the ITCH/NUMB complex

doi: 10.1038/s42003-018-0275-4

Figure Lengend Snippet: MEP50/PRMT5 complex-mediated GLI1 stabilisation enhances Gli transcriptional activity and HH signalling pathway activation induces PRMT5 and MEP50 expression. a Gli transcriptional activity in PRMT5 or MEP50 knockdown cells. siMEP50-m2 and siPRMT5-m2 siRNAs were stably expressed by recombinant retroviruses. A multimerized Gli-binding site luciferase reporter plasmid and phRL-TK control reporter plasmid were transfected into C3H10T1/2 cells. After 24 h of incubation, 300 nM SAG was applied for 24 h, and then luciferase assays were performed. b qRT-PCR analysis of Ptch1 , Bcl2 , and Foxm1 expression in C3H10T1/2 cells with MEP50 knockdown or PRMT5 knockdown and treated with 300 nM SAG for the indicated times. siMEP50-m2 and siPRMT5-m2 siRNAs were stably expressed by recombinant retroviruses. c Gli transcriptional activity in HA-PRMT5 or Myc-MEP50-expressing cells. HA-PRMT5, HA-PRMT5 G367A/R368A, or Myc-MEP50 and a multimerized Gli-binding site luciferase reporter plasmid and phRL-TK control reporter plasmid were transfected into C3H10T1/2 cells. After 24 h of incubation, 300 nM SAG was applied for 24 h, and then luciferase assays were performed. d qRT-PCR analysis of Ptch1 , Bcl2 , and Foxm1 expression in HA-PRMT5 or Myc-MEP50-expressing C3H10T1/2 cells. HA-PRMT5, HA-PRMT5 G367A/R368A, or Myc-MEP50 plasmids were transfected into C3H10T1/2 cells. After 24 h of incubation, cells were separated equally, and DMSO (−) or 300 nM SAG (+) were applied for 24 h. Protein levels are shown in Supplementary Fig. . e and f qRT-PCR analysis of PRMT5 ( e ) and MEP50 ( f ) mRNA expression in C3H10T1/2 cells after 24 h of treatment with 300 nM SAG. In a – c , data represent one of two independent experiments with similar results. In e and f , data represent one of three independent experiments with similar results. The source data is shown in Supplementary Data

Article Snippet: The TaqMan gene expression assays used for mouse samples were Ptch1 (Mm01306905_m1), Gli1 (Mm00494645_m1), Foxm1 (Mm00514924_m1), Bcl2 (Mm00477631_m1), Mep50 (Mm01296589_g1), and Prmt5 (Mm00550472_m1).

Techniques: Activity Assay, Activation Assay, Expressing, Knockdown, Stable Transfection, Recombinant, Binding Assay, Luciferase, Plasmid Preparation, Control, Transfection, Incubation, Quantitative RT-PCR

Journal: Communications Biology

Article Title: MEP50/PRMT5-mediated methylation activates GLI1 in Hedgehog signalling through inhibition of ubiquitination by the ITCH/NUMB complex

doi: 10.1038/s42003-018-0275-4

Figure Lengend Snippet: MEP50/PRMT5 complex induces GLI1 methylation. a , b Methylation of GLI1 in MEP50- ( a ) or PRMT5- ( b ) knockdown C3H10T1/2 cells. siMEP50-m2 and siPRMT5-m2 siRNAs were stably expressed by recombinant retroviruses. Cells transfected with FLAG-GLI1 were cultured for 24 h, followed by treatment with 300 nM SAG for 24 h. Methylated GLI1 was detected by immunoprecipitation with an anti-FLAG antibody followed by immunoblot with anti-SYM11 antibody. c In vitro methylation assays to determine the region including methylated arginine residues in GLI1 deletion mutants. HA-PRMT5 expression plasmid was transfected into HEK293T cells. At 48 h after transfection, the cells were lysed, and HA-PRMT5 was immunoprecipitated using an anti-HA (3F10) antibody. GST-GLI1 deletion mutants coupled to glutathione sepharose were incubated with immunoprecipitated HA-PRMT5 from HEK293T cells. Upper panel represents the methylated GST-GLI1 deletion mutant. Lower panel represents 20% input of GST-GLI1 deletion mutants detected by CBB R-250 staining. HA-PRMT5 expressed in 10% of total lysate used for immunoprecipitation is shown in the right panel. d In vitro methylation assays to determine methylation sites in GLI1 using amino acid substitutions (arginine to lysine) of candidate methylation sites. In vitro methylation assays were performed as described in ( c ). Upper panel represents methylated GST-GLI1 mutants. Lower panel represents 20% input of GST-GLI1 mutants detected by CBB R-250 staining. Underlined text denotes highly conserved residues among mammals, as shown in Supplementary Fig. . In c , data represent one of three independent experiments with similar results. In a and d , data represent one of twice independent experiments with similar results. Unprocessed original scans of blots are shown in Supplementary Fig.

Article Snippet: The TaqMan gene expression assays used for mouse samples were Ptch1 (Mm01306905_m1), Gli1 (Mm00494645_m1), Foxm1 (Mm00514924_m1), Bcl2 (Mm00477631_m1), Mep50 (Mm01296589_g1), and Prmt5 (Mm00550472_m1).

Techniques: Methylation, Knockdown, Stable Transfection, Recombinant, Transfection, Cell Culture, Immunoprecipitation, Western Blot, In Vitro, Expressing, Plasmid Preparation, Incubation, Mutagenesis, Staining

Journal: Communications Biology

Article Title: MEP50/PRMT5-mediated methylation activates GLI1 in Hedgehog signalling through inhibition of ubiquitination by the ITCH/NUMB complex

doi: 10.1038/s42003-018-0275-4

Figure Lengend Snippet: MEP50/PRMT5 complex-mediated GLI1 methylation inhibits the interaction of GLI1 with its E3 ligase complex, ITCH/NUMB, resulting in GLI1 stabilisation. a Interaction of GLI1 and endogenous ITCH or NUMB from stably PRMT5-knockdown or MEP50-knockdown C3H10T1/2 cells. siMEP50-m2 and siPRMT5-m2 siRNAs were stably expressed by recombinant retroviruses. MG132 (50 μM) was applied for 4 h before harvesting. b Interaction of GLI1 mutants with endogenous ITCH or NUMB in C3H10T1/2 cells. The cells were transfected as indicated. At 48 h post-transfection, 50 μM MG132 was applied for 4 h, and then the cells were lysed and subjected to immunoprecipitation with an anti-HA antibody, followed by immunoblotting with antibodies against the indicated proteins. c In vivo ubiquitination of HA-GLI1-RK mutants. Cells were transfected and cultured for 24 h, followed by treatment with 50 µM MG132 for 4 h before harvesting. Ubiquitinated GLI1 was detected by immuoprecipitation with an anti-HA (3F10) antibody and immunoblotting with anti-FLAG (upper panel) or anti-HA (lower panel) antibodies. The asterisk denotes non-specific bands. d Schematic diagram of the mechanism of PRMT5/MEP50-mediated GLI1 stabilisation. When the HH signalling pathway inactivates, the ITCH/NUMB E3 ligase complex binds to and ubiquitinates GLI1 for proteasomal degradation. In turn, under HH signalling pathway activation, the MEP50/PRMT5 complex methylates GLI1 to dissociate the ITCH/NUMB complex from GLI1, resulting in GLI1 stabilisation. Unprocessed original scans of blots are shown in Supplementary Fig.

Article Snippet: The TaqMan gene expression assays used for mouse samples were Ptch1 (Mm01306905_m1), Gli1 (Mm00494645_m1), Foxm1 (Mm00514924_m1), Bcl2 (Mm00477631_m1), Mep50 (Mm01296589_g1), and Prmt5 (Mm00550472_m1).

Techniques: Methylation, Stable Transfection, Knockdown, Recombinant, Transfection, Immunoprecipitation, Western Blot, In Vivo, Ubiquitin Proteomics, Cell Culture, Activation Assay

Journal: Communications Biology

Article Title: MEP50/PRMT5-mediated methylation activates GLI1 in Hedgehog signalling through inhibition of ubiquitination by the ITCH/NUMB complex

doi: 10.1038/s42003-018-0275-4

Figure Lengend Snippet: PRMT5 and MEP50 expression is upregulated in HH pathway-activated cancers, and PRMT5 inhibition is a potential therapeutic strategy for such cancers. a , b Immunoblot analysis of endogenous GLI1 in H146 and AGS cells stably expressing PRMT5 ( a ) or MEP50 ( b ) siRNAs. c Immunoblot analysis of endogenous GLI1 in H146 cells stably expressing GLI1 siRNA. In a – c , siRNAs were stably expressed via recombinant retroviruses. d Growth curves of PRMT5, MEP50, and GLI1-knockdown H146 SCLC cells. Results are shown in the mean ± s.d. of triplicate experiments. e A quantitative colony formation assay was performed by plating cells at a density of 1 × 10 4 cells in a six-well plate and incubating them for 14 days. Surviving colonies were counted and represented as the mean ± s.d. of three independent wells. In d and e , siMEP50, siPRMT5, or siGLI1 was stably expressed via recombinant retrovirus in H146 cells. f , g IC 50 values of cyclopamine in PRMT5-knockdown or MEP50-knockdown AGS cells. siRNAs were stably expressed via recombinant retroviruses. Cell viability ( e ) is shown as the mean ± s.d. n = 4. IC 50 values of cyclopamine are shown in g . h – j Upregulated expression of PRMT5 , MEP50 , and GLI1 target genes in small cell lung carcinoma ( h ), gastric adenocarcinoma ( i ), and skin basal cell carcinoma ( j ) from the ONCOMINE database ( https://www.oncomine.org/ ). The threshold of data was p ≤ 0.05. Each boxplot shows the log maximum, minimum, and median signal intensity of each mRNA from the corresponding expression array. Bold lines on each boxplot define the median value. P -values and sample numbers are indicated in each panel. Unprocessed original scans of blots are shown in Supplementary Fig. . Source data of d – f is shown in Supplementary Data

Article Snippet: The TaqMan gene expression assays used for mouse samples were Ptch1 (Mm01306905_m1), Gli1 (Mm00494645_m1), Foxm1 (Mm00514924_m1), Bcl2 (Mm00477631_m1), Mep50 (Mm01296589_g1), and Prmt5 (Mm00550472_m1).

Techniques: Expressing, Inhibition, Western Blot, Stable Transfection, Recombinant, Knockdown, Colony Assay

Journal: Communications Biology

Article Title: MEP50/PRMT5-mediated methylation activates GLI1 in Hedgehog signalling through inhibition of ubiquitination by the ITCH/NUMB complex

doi: 10.1038/s42003-018-0275-4

Figure Lengend Snippet: Primer sequences used for PRMT5 and MEP50 cloning

Article Snippet: The TaqMan gene expression assays used for mouse samples were Ptch1 (Mm01306905_m1), Gli1 (Mm00494645_m1), Foxm1 (Mm00514924_m1), Bcl2 (Mm00477631_m1), Mep50 (Mm01296589_g1), and Prmt5 (Mm00550472_m1).

Techniques: Sequencing

Journal: bioRxiv

Article Title: Regulation of transcription termination by FUS and TDP-43

doi: 10.1101/788778

Figure Lengend Snippet: A . Immunoprecipitation (IP) with the indicated antibodies from HEK293 whole cell lysate (WCL), followed by western blotting with the indicated antibodies. IP with IgG was used as a negative control. The blots indicate that POLR2A, SMN, FUS, TDP-43, PRMT5 and the termination factors SETX and XRN2 interact directly or indirectly with each other . B . A summary of the interactions detected by co-IP experiments. All of these interactions, except the SMN-XRN2 and SMN-TDP-43 interactions, have been shown by various published experiments to be direct ( , , ).

Article Snippet: 8WG16 antibody against unphosphorylated CTD repeats of POLR2A was prepared in the lab. Commercial antibodies were as follows: HA (Sigma, mAb H9658); PRMT5 (Upstate, pAb C7-405, Santa Cruz, mAb sc-22132); SMN (Santa Cruz, pAb H-195); SETX for ChIP and IP (Novus Biologicals, pAb NB100-57543) and for western blots (Bethyl Lab, pAb A301-104A); XRN2 (Santa Cruz, pAb sc-99237); FUS (Santa Cruz, mAb sc-47711); TDP-43 (Bethyl lab, pAb A303-233A); POLR2A N20 (Santa Cruz, pAb sc-899); POLR2A 4H8 (Abcam, mAb ab5408); gammaH2Ax (Millipore, 05-636); H2Ax (Millipore, 07-627); Tubulin (Sigma, mAb T8328); GFP (Abcam, pAb 290); IgG negative controls for ChIP and IP (Millipore, pAb 12-370). α-amanitin was purchased from Sigma (23109-05-9).

Techniques: Immunoprecipitation, Western Blot, Negative Control, Co-Immunoprecipitation Assay

Journal: bioRxiv

Article Title: Regulation of transcription termination by FUS and TDP-43

doi: 10.1101/788778

Figure Lengend Snippet: A . IP with 8WG16 antibodies for POLR2A from HEK293 WCL upon stably knocking down PRMT5 or SMN, with the knock-down of GFP as negative control. Western blots were performed with the indicated antibodies. The knock-down of PRMT5 or SMN caused a reduction of FUS and TDP-43 interaction with RNAPII. B . IP with the indicated antibodies from HEK293 WCL, upon knocking out of SMN with the CRISPR/Cas9 system or using scrambled guide RNA as negative control. Western blots were performed with the indicated antibodies to show that the SMN knock-out leads to a loss of interaction of TDP-43 and SETX with POLR2A. IP with IgG as negative control. C, D . Quantifications of ChIP in HEK293 cells as FUS/POLR2A ( C ) or TDP-43/POLR2A ( D ) ratio to show the relative effects of knocking down PRMT5 or SMN. Error bars denote biological replicate s.e.m. (n=3).

Article Snippet: 8WG16 antibody against unphosphorylated CTD repeats of POLR2A was prepared in the lab. Commercial antibodies were as follows: HA (Sigma, mAb H9658); PRMT5 (Upstate, pAb C7-405, Santa Cruz, mAb sc-22132); SMN (Santa Cruz, pAb H-195); SETX for ChIP and IP (Novus Biologicals, pAb NB100-57543) and for western blots (Bethyl Lab, pAb A301-104A); XRN2 (Santa Cruz, pAb sc-99237); FUS (Santa Cruz, mAb sc-47711); TDP-43 (Bethyl lab, pAb A303-233A); POLR2A N20 (Santa Cruz, pAb sc-899); POLR2A 4H8 (Abcam, mAb ab5408); gammaH2Ax (Millipore, 05-636); H2Ax (Millipore, 07-627); Tubulin (Sigma, mAb T8328); GFP (Abcam, pAb 290); IgG negative controls for ChIP and IP (Millipore, pAb 12-370). α-amanitin was purchased from Sigma (23109-05-9).

Techniques: Stable Transfection, Knockdown, Negative Control, Western Blot, CRISPR, Knock-Out

Journal: Cellular signalling

Article Title: Inhibition of PRMT5 suppresses osteoclast differentiation and partially protects against ovariectomy-induced bone loss through downregulation of CXCL10 and RSAD2.

doi: 10.1016/j.cellsig.2017.03.004

Figure Lengend Snippet: Fig. 1. PRMT5 is upregulated during osteoclast differentiation, and PRMT5 knockdown inhibits osteoclast differentiation. (A) BMMs were cultured in the presence of M-CSF (30 ng/mL) and RANKL (100 ng/mL) and were collected at the indicated time points to analyze PRMT5 and c-fos protein expression. (B–C) PRMT5 protein expression in BMMs cells was assessed by western blotting. GAPDH was used as a loading control. Band densities were quantified and normalized to the control. Data are presented as mean ± SD of three independent experiments. *P b 0.05. (D–E) BMMs were seeded in 96-well plates at a density of 1.5 × 104 cells per well, transfected with PRMT5 siRNA and cultured in the presence of RANKL (100 ng/mL) and M-CSF (30 ng/mL) for 5 days. TRAP staining was performed, and TRAP-positive cells with three or more nuclei were counted using a microscope. Data are presented as mean ± SD of three independent experiments. *P b 0.05, **P b 0.01 versus vehicle.

Article Snippet: The PRMT5 inhibitor EPZ015666 (EPZ) was obtained from Selleck Chemicals (Houston, TX, USA).

Techniques: Knockdown, Cell Culture, Expressing, Western Blot, Control, Transfection, Staining, Microscopy

Journal: Cellular signalling

Article Title: Inhibition of PRMT5 suppresses osteoclast differentiation and partially protects against ovariectomy-induced bone loss through downregulation of CXCL10 and RSAD2.

doi: 10.1016/j.cellsig.2017.03.004

Figure Lengend Snippet: Fig. 8. EPZ inhibits osteoclastogenesis partially through downregulation of CXCL10 and RSAD2. (A) BMMs were treated with the indicated concentrations of EPZ in the presence of 100 ng/mL RANKL and 30 ng/mL M-CSF for 2 or 4 days. The medium was collected, and the CXCL10 protein level was assessed by ELISA. Data are presented as mean ± SD of three independent experiments. (B–C) BMMs were incubated with RANKL (100 ng/mL) and M-CSF (30 ng/mL), and then treated with EPZ (10 μM) and CXCL10 (10 ng/mL). Five days later, cells were fixed for TRAP staining. Representative images are shown, and TRAP-positive multinucleated osteoclasts (≥3 nuclei) were counted. (D, E) BMMs were treated with EPZ (10 μM) in the presence of M-CSF for 3 days. Expression of PRMT5 and RSAD2 protein was assessed by western blotting. GAPDH was used as a loading control. Band densities were quantified and normalized to the control. Data are presented as mean ± SD of three independent experiments. (F, G) BMMs were seeded in 96-well plates at a density of 1.5 × 104

Article Snippet: The PRMT5 inhibitor EPZ015666 (EPZ) was obtained from Selleck Chemicals (Houston, TX, USA).

Techniques: Enzyme-linked Immunosorbent Assay, Incubation, Staining, Expressing, Western Blot, Control

Journal: Journal of Biomedical Science

Article Title: Cardiac-specific overexpression of PRMT5 exacerbates pressure overload-induced hypertrophy and heart failure

doi: 10.1186/s12929-025-01162-6

Figure Lengend Snippet: Cardiac-specific overexpression of PRMT5 accelerates pressure overload-induced cardiac systolic dysfunction. a A schematic diagram of the transgene to create mice with cardiac-specific Prmt5 overexpression (PRMT5-TG). b Images of WT and PRMT5-TG mice littermates at 10 weeks of age. Scale bar: 20 µm. c Cardiac PRMT5 overexpression confirmed using Western blotting. d Echocardiographic analysis images of PRMT5-TG mice performed 4 weeks after TAC surgery. e , f Fractional shorting ( e ) and ejection fraction ( f ) calculated from M-mode echocardiography. Values are presented as mean ± SD (n = 6–8 mice/group). Data are analyzed using two-way ANOVA, followed by Tukey’s multiple comparison test. A p < 0.05 is considered statistically significant. *** p < 0.001

Article Snippet: For Western blotting analysis, the primary antibodies used in this study were an anti-DDDDK-tag mAb (Cat#M185-3L, MBL Life Science), anti-HA-tag mAb (Cat#M132-3, MBL Life Science), anti-Myc-tag mAb (Cat#M192-3, MBL Life Science), anti-PRMT5 antibody (Cat#07-405, Merck, Tokyo, Japan), Symmetric Di-Methyl Arginine Motif Rabbit mAb (#13222, Cell Signaling Technology, Tokyo, Japan), anti-β-actin mouse monoclonal clone AC-15 IgG (Cat#A1978, Sigma-Aldrich), anti-GAPDH rabbit polyclonal antibody (10494-1-AP, Proteintech), anti-histone-H3 polyclonal antibody (17168-1-AP, Proteintech), acetyl-Histone H3 (Lys9) rabbit mAb (#9649, Cell Signaling Technology), rabbit polyclonal anti-acetyl-H3 (K14) antibody (#7627, Cell Signaling Technology), rabbit anti-dimethyl-histone H3 (Arg2) antibody (Cat#07-585, Merck), rabbit polyclonal anti-H4R3me2s antibody (Cat#AB_2793544, Active motif, Tokyo, Japan), and anti-Histone H3 (acetyl K122) antibody (ab33309, Abcam, Cambridge, UK).

Techniques: Over Expression, Western Blot, Comparison

Journal: Journal of Biomedical Science

Article Title: Cardiac-specific overexpression of PRMT5 exacerbates pressure overload-induced hypertrophy and heart failure

doi: 10.1186/s12929-025-01162-6

Figure Lengend Snippet: Cardiac overexpression of PRMT5 promotes pressure overload-induced cardiac hypertrophy. a Representative photos of the hearts extracted from the mice. b Histological analysis of the heart tissues. Scale bars: 2 mm. c Heart weight/body weight comparisons at 4 weeks after TAC surgery. Values are presented as mean ± SD (n = 6–8 mice/group) d Cardiac overexpression of PRMT5 promotes the increases in lung weight after TAC surgery. e , f Histological analysis results of PRMT5-TG mice at 4 weeks after TAC surgery. Representative images of HE-stained sections of the mice hearts. Cross-sectional myocardial cell diameter measurement ( e ). Representative images of MT-stained perivascular and interstitial fibrosis area of the LV myocardium of the mice. Perivascular and interstitial fibrotic area measurements ( f ). Values are presented as mean ± SD (n = 5 mice/group). Scale bars: 20 µm ( e ), 50 µm (Perivascular) and 100 µm (interstitial) ( f ). g Hypertrophic gene expression is increased by cardiac overexpression of PRMT5. Values are expressed as mean ± SD (n = 7–8 mice/group). Data are analyzed using two-way ANOVA, followed by Tukey’s multiple comparison test. A p < 0.05 is considered statistically significant. * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: For Western blotting analysis, the primary antibodies used in this study were an anti-DDDDK-tag mAb (Cat#M185-3L, MBL Life Science), anti-HA-tag mAb (Cat#M132-3, MBL Life Science), anti-Myc-tag mAb (Cat#M192-3, MBL Life Science), anti-PRMT5 antibody (Cat#07-405, Merck, Tokyo, Japan), Symmetric Di-Methyl Arginine Motif Rabbit mAb (#13222, Cell Signaling Technology, Tokyo, Japan), anti-β-actin mouse monoclonal clone AC-15 IgG (Cat#A1978, Sigma-Aldrich), anti-GAPDH rabbit polyclonal antibody (10494-1-AP, Proteintech), anti-histone-H3 polyclonal antibody (17168-1-AP, Proteintech), acetyl-Histone H3 (Lys9) rabbit mAb (#9649, Cell Signaling Technology), rabbit polyclonal anti-acetyl-H3 (K14) antibody (#7627, Cell Signaling Technology), rabbit anti-dimethyl-histone H3 (Arg2) antibody (Cat#07-585, Merck), rabbit polyclonal anti-H4R3me2s antibody (Cat#AB_2793544, Active motif, Tokyo, Japan), and anti-Histone H3 (acetyl K122) antibody (ab33309, Abcam, Cambridge, UK).

Techniques: Over Expression, Staining, Gene Expression, Comparison

Journal: Journal of Biomedical Science

Article Title: Cardiac-specific overexpression of PRMT5 exacerbates pressure overload-induced hypertrophy and heart failure

doi: 10.1186/s12929-025-01162-6

Figure Lengend Snippet: Pharmacological inhibition and knockdown of PRMT5 suppresses phenylephrine (PE)-induced hypertrophic responses in cultured cardiomyocytes. a Immunostaining images for α-actinin to determine the cell surface area of cardiomyocytes, quantified using ImageJ software. Scale bars: 20 µm. b Hypertrophic gene expression levels of Nppa and Nppb , quantified by qRT-PCR. c Primary cultured cardiomyocytes were transfected with siRNA (siControl or siPrmt5) and then stimulated with or without PE (30 µM). Prmt5 knockdown is confirmed by qRT-PCR and WB. The cell surface area was quantified using ImageJ software. Scale bars: 20 µm. d PE-induced Nppa and Nppb gene expression as quantified by qRT-PCR and Western blotting. Values are presented as mean ± SD (n = 4–5). Data are analyzed using one-way ANOVA, followed by Dunnett’s multiple comparison tests versus the PE-treated group. * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: For Western blotting analysis, the primary antibodies used in this study were an anti-DDDDK-tag mAb (Cat#M185-3L, MBL Life Science), anti-HA-tag mAb (Cat#M132-3, MBL Life Science), anti-Myc-tag mAb (Cat#M192-3, MBL Life Science), anti-PRMT5 antibody (Cat#07-405, Merck, Tokyo, Japan), Symmetric Di-Methyl Arginine Motif Rabbit mAb (#13222, Cell Signaling Technology, Tokyo, Japan), anti-β-actin mouse monoclonal clone AC-15 IgG (Cat#A1978, Sigma-Aldrich), anti-GAPDH rabbit polyclonal antibody (10494-1-AP, Proteintech), anti-histone-H3 polyclonal antibody (17168-1-AP, Proteintech), acetyl-Histone H3 (Lys9) rabbit mAb (#9649, Cell Signaling Technology), rabbit polyclonal anti-acetyl-H3 (K14) antibody (#7627, Cell Signaling Technology), rabbit anti-dimethyl-histone H3 (Arg2) antibody (Cat#07-585, Merck), rabbit polyclonal anti-H4R3me2s antibody (Cat#AB_2793544, Active motif, Tokyo, Japan), and anti-Histone H3 (acetyl K122) antibody (ab33309, Abcam, Cambridge, UK).

Techniques: Inhibition, Knockdown, Cell Culture, Immunostaining, Software, Gene Expression, Quantitative RT-PCR, Transfection, Western Blot, Comparison

Journal: Journal of Biomedical Science

Article Title: Cardiac-specific overexpression of PRMT5 exacerbates pressure overload-induced hypertrophy and heart failure

doi: 10.1186/s12929-025-01162-6

Figure Lengend Snippet: PRMT5 modifies histone methylation and acetylation in hearts and cultured cardiomyocytes. a Acid extracts from mouse hearts of WT and PRMT5 transgenic (PRMT5-TG) mice were applied to Western blotting analysis. Western blotting was performed using the indicated antibodies. b WT and PRMT5-TG mice were subjected to the TAC surgery. The samples prepared from these hearts were used for Western blotting. c Western blotting was performed using acid extracts from mouse hearts of sham and TAC mice treated with or without the PRMT5 inhibitor EPZ015666. Values are presented as mean ± SD (n = 4 mice). d Cultured cardiomyocytes were treated with EPZ015666 in the presence or absence of PE. Values are presented as mean ± SD (n = 3). e HAT activity in hearts was measured using a fluorescent-based method. A p300 HAT inhibitor, C646, was added to the protein extracts from the hearts of PRMT5-TG mice. Values are presented as mean ± SD (n = 10 mice/group). f Immunostaining images for α-actinin to determine the cell surface area of cardiomyocytes, quantified using ImageJ software. Scale bars: 20 µm. Values are presented as mean ± SD (n = 4). Data are analyzed using one-way ANOVA, followed by Tukey’s multiple comparison ( c , e , f ) or Dunnett’s multiple comparison tests versus the PE-treated group ( d ). * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: For Western blotting analysis, the primary antibodies used in this study were an anti-DDDDK-tag mAb (Cat#M185-3L, MBL Life Science), anti-HA-tag mAb (Cat#M132-3, MBL Life Science), anti-Myc-tag mAb (Cat#M192-3, MBL Life Science), anti-PRMT5 antibody (Cat#07-405, Merck, Tokyo, Japan), Symmetric Di-Methyl Arginine Motif Rabbit mAb (#13222, Cell Signaling Technology, Tokyo, Japan), anti-β-actin mouse monoclonal clone AC-15 IgG (Cat#A1978, Sigma-Aldrich), anti-GAPDH rabbit polyclonal antibody (10494-1-AP, Proteintech), anti-histone-H3 polyclonal antibody (17168-1-AP, Proteintech), acetyl-Histone H3 (Lys9) rabbit mAb (#9649, Cell Signaling Technology), rabbit polyclonal anti-acetyl-H3 (K14) antibody (#7627, Cell Signaling Technology), rabbit anti-dimethyl-histone H3 (Arg2) antibody (Cat#07-585, Merck), rabbit polyclonal anti-H4R3me2s antibody (Cat#AB_2793544, Active motif, Tokyo, Japan), and anti-Histone H3 (acetyl K122) antibody (ab33309, Abcam, Cambridge, UK).

Techniques: Methylation, Cell Culture, Transgenic Assay, Western Blot, Activity Assay, Immunostaining, Software, Comparison

Journal: Journal of Biomedical Science

Article Title: Cardiac-specific overexpression of PRMT5 exacerbates pressure overload-induced hypertrophy and heart failure

doi: 10.1186/s12929-025-01162-6

Figure Lengend Snippet: PRMT5 methylates p300 at R200. a The plasmids of WT PRMT5 and the deletion mutant lacking enzymatic activity of PRMT5 (ΔPRMT5) were transfected into HEK293T cells. Immunoprecipitation of p300 and Western blotting was performed. b GST pull-down assay was performed with GST-fusioned aa1-450, aa1514-1922, and aa1817-2160 of p300 mutants extracted from E. coli . [ 35 S]radiolabeled PRMT5 was obtained using an in vitro translation system. The arrows show GST fusion proteins. c PRMT5 and aa1-450, aa1514-1922, and aa1817-2160 of p300 mutants were mixed in reaction solution with [ 14 C]radiolabeled S-adenosyl methionine (SAM). The proteins were analyzed using SDS-PAGE and scanned using BAS2000. d PRMT5 and R200K, R202K, and R237K of p300 aa1-450 point mutants were mixed in reaction solution with [ 14 C]radiolabeled SAM, and an in vitro methylation assay was performed

Article Snippet: For Western blotting analysis, the primary antibodies used in this study were an anti-DDDDK-tag mAb (Cat#M185-3L, MBL Life Science), anti-HA-tag mAb (Cat#M132-3, MBL Life Science), anti-Myc-tag mAb (Cat#M192-3, MBL Life Science), anti-PRMT5 antibody (Cat#07-405, Merck, Tokyo, Japan), Symmetric Di-Methyl Arginine Motif Rabbit mAb (#13222, Cell Signaling Technology, Tokyo, Japan), anti-β-actin mouse monoclonal clone AC-15 IgG (Cat#A1978, Sigma-Aldrich), anti-GAPDH rabbit polyclonal antibody (10494-1-AP, Proteintech), anti-histone-H3 polyclonal antibody (17168-1-AP, Proteintech), acetyl-Histone H3 (Lys9) rabbit mAb (#9649, Cell Signaling Technology), rabbit polyclonal anti-acetyl-H3 (K14) antibody (#7627, Cell Signaling Technology), rabbit anti-dimethyl-histone H3 (Arg2) antibody (Cat#07-585, Merck), rabbit polyclonal anti-H4R3me2s antibody (Cat#AB_2793544, Active motif, Tokyo, Japan), and anti-Histone H3 (acetyl K122) antibody (ab33309, Abcam, Cambridge, UK).

Techniques: Mutagenesis, Activity Assay, Transfection, Immunoprecipitation, Western Blot, Pull Down Assay, In Vitro, SDS Page, Methylation

Journal: Journal of Biomedical Science

Article Title: Cardiac-specific overexpression of PRMT5 exacerbates pressure overload-induced hypertrophy and heart failure

doi: 10.1186/s12929-025-01162-6

Figure Lengend Snippet: PRMT5-mediated p300 arginine methylation of R200 is required for p300 histone acetyltransferase activity. a The plasmids encoding p300 WT or p300 R200K point mutant and PRMT5 were transfected to HEK293T cells. Immunoprecipitation by anti-HA antibody followed by Western blotting was performed. The band density was measured using ImageJ software. Values are presented as mean ± SD (n = 3). b p300 WT and p300 R200K point mutant were purified using anti-HA-tag agarose beads from HEK293T cells co-transfected with or without PRMT5. Purified p300 was incubated with recombinant histone H3 peptide and [ 3 H]-labeled acetyl-CoA. The histone H3 peptides were isolated, and radioactivity was measured using a liquid scintillation counter. Values are presented as mean ± SD (n = 6). Data are analyzed using two-way ANOVA, followed by Tukey’s multiple comparison test. p < 0.05 was considered statistically significant. * p < 0.05, *** p < 0.001

Article Snippet: For Western blotting analysis, the primary antibodies used in this study were an anti-DDDDK-tag mAb (Cat#M185-3L, MBL Life Science), anti-HA-tag mAb (Cat#M132-3, MBL Life Science), anti-Myc-tag mAb (Cat#M192-3, MBL Life Science), anti-PRMT5 antibody (Cat#07-405, Merck, Tokyo, Japan), Symmetric Di-Methyl Arginine Motif Rabbit mAb (#13222, Cell Signaling Technology, Tokyo, Japan), anti-β-actin mouse monoclonal clone AC-15 IgG (Cat#A1978, Sigma-Aldrich), anti-GAPDH rabbit polyclonal antibody (10494-1-AP, Proteintech), anti-histone-H3 polyclonal antibody (17168-1-AP, Proteintech), acetyl-Histone H3 (Lys9) rabbit mAb (#9649, Cell Signaling Technology), rabbit polyclonal anti-acetyl-H3 (K14) antibody (#7627, Cell Signaling Technology), rabbit anti-dimethyl-histone H3 (Arg2) antibody (Cat#07-585, Merck), rabbit polyclonal anti-H4R3me2s antibody (Cat#AB_2793544, Active motif, Tokyo, Japan), and anti-Histone H3 (acetyl K122) antibody (ab33309, Abcam, Cambridge, UK).

Techniques: Methylation, Activity Assay, Mutagenesis, Transfection, Immunoprecipitation, Western Blot, Software, Purification, Incubation, Recombinant, Labeling, Isolation, Radioactivity, Comparison

Journal: Journal of Biomedical Science

Article Title: Cardiac-specific overexpression of PRMT5 exacerbates pressure overload-induced hypertrophy and heart failure

doi: 10.1186/s12929-025-01162-6

Figure Lengend Snippet: Graphical abstract. The gain-of-function of PRMT5 accelerates pressure overload-induced cardiomyocyte hypertrophy and heart failure. PRMT5 methylates p300 at R200 and regulates p300 HAT activity, which is essential for histone acetylation during the development of cardiomyocyte hypertrophy

Article Snippet: For Western blotting analysis, the primary antibodies used in this study were an anti-DDDDK-tag mAb (Cat#M185-3L, MBL Life Science), anti-HA-tag mAb (Cat#M132-3, MBL Life Science), anti-Myc-tag mAb (Cat#M192-3, MBL Life Science), anti-PRMT5 antibody (Cat#07-405, Merck, Tokyo, Japan), Symmetric Di-Methyl Arginine Motif Rabbit mAb (#13222, Cell Signaling Technology, Tokyo, Japan), anti-β-actin mouse monoclonal clone AC-15 IgG (Cat#A1978, Sigma-Aldrich), anti-GAPDH rabbit polyclonal antibody (10494-1-AP, Proteintech), anti-histone-H3 polyclonal antibody (17168-1-AP, Proteintech), acetyl-Histone H3 (Lys9) rabbit mAb (#9649, Cell Signaling Technology), rabbit polyclonal anti-acetyl-H3 (K14) antibody (#7627, Cell Signaling Technology), rabbit anti-dimethyl-histone H3 (Arg2) antibody (Cat#07-585, Merck), rabbit polyclonal anti-H4R3me2s antibody (Cat#AB_2793544, Active motif, Tokyo, Japan), and anti-Histone H3 (acetyl K122) antibody (ab33309, Abcam, Cambridge, UK).

Techniques: Activity Assay