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: Oncogene

Article Title: PRMT5 methylating SMAD4 activates TGF-β signaling and promotes colorectal cancer metastasis.

doi: 10.1038/s41388-023-02674-x

Figure Lengend Snippet: Fig. 5 Targeting the methylation of SMAD4 at R361 inhibits CRC metastasis in vivo. A, B 2 × 106 luciferase-expressing LoVo cells of different genotypes, shNC, PRMT5 sh#1 and sh#2, were injected intrasplenic to BALB/c nude mice. After 6 weeks, bioluminescence was detected, then livers were dissected, and metastatic tumors were weighed. Representative images (A) and quantitative analysis of metastatic tumor weight (B) (*p < 0.05, **p < 0.01, t-test). C IHC staining of PRMT5 and SMAD4 R361me2s in tumor tissue. D, E 2 × 106 luciferase-expressing cells of different genotypes, WT, R361K mutant, PRMT5 overexpressing and PRMT5 overexpressing R361K mutant, were injected intrasplenic into BALB/c nude mice. After 3 weeks of tumor cell injection, half of the mice injected with WT and R361K cells were treated with GSK3326595 (40 mg/kg) every 3 days. After another 3 weeks, bioluminescence was detected, then livers were dissected, and metastatic tumors were weighed. Representative images (D) and quantitative analysis of metastatic tumor weight (E) (*p < 0.05, **p < 0.01, t-test).

Article Snippet: His & Flag-tagged PRMT5 recombinant protein (Sino Biological, Cat: 11074- H18H, China), GST-tagged fusion protein Smad4 (Proteintech, Cat No: Ag0299, USA), and MEP50 recombinant protein (Abnova, H00079084-P01, China) pure proteins were purchased.

Techniques: Methylation, In Vivo, Luciferase, Expressing, Injection, Immunohistochemistry, Mutagenesis

Journal:

Article Title: Methylation of Histone H3 and H4 by PRMT5 Regulates Ribosomal RNA Gene Transcription

doi: 10.1002/jcb.22432

Figure Lengend Snippet: Association of PRMT5 with rRNA promoter is reduced in EBV transformed B-cells. A: PRMT5 level was monitored by Western blot analysis in EBV transformed cells (LCLs), EBV-infected and resting B cells. B: Resting B-cells and exponentially growing EBV transformed cells (LCL1, LCL2) were cross-linked with formaldehyde and chromatin from these cells was immunoprecipitated with anti-PRMT5 antibody. The pulled down DNA was either left uncut (U) or digested with Hpa II (H) or Msp I (M). rRNA promoter was amplified from all three sets of precipitated and digested DNA and analyzed by semi-quantitative PCR. The bar diagram represents association of PRMT5 with unmethylated and methylated rRNA promoter (as described in Fig. 2F).

Article Snippet: PRMT5 siRNA AND ITS TRANSFECTION HeLa cells were transfected at 70% confluency with 100 nM PRMTS siRNA (Santa Cruz, SC37022) or the control scrambled siRNA (Santa Cruz, SC37007) using Lipofectamine 2000.

Techniques: Transformation Assay, Western Blot, Infection, Immunoprecipitation, Amplification, Real-time Polymerase Chain Reaction, Methylation

Journal:

Article Title: Methylation of Histone H3 and H4 by PRMT5 Regulates Ribosomal RNA Gene Transcription

doi: 10.1002/jcb.22432

Figure Lengend Snippet: PRMT5 expression remains unaltered in serum starved and serum replenished HeLa cells. A: Exponentially growing HeLa cells were serum starved (0%) for 72 h (−FBS) and then allowed to grow in presence of 10% serum for 3 h. 47S rRNA synthesis in these cells was compared by real time PCR and normalized to β-actin. B: Whole cell extracts from HeLa cells (−FBS and +FBS) were subjected to Western blot analysis with anti-PRMT5 antibody. The blot was reprobed with anti-GAPDH antibody for protein normalization. C: PRMT5 co-localizes with nucleolin in HeLa cell nucleolus. HeLa cells were stained with TRITC tagged mouse monoclonal antibody against nucleolin and with FITC-tagged rabbit polyclonal antibody against PRMT5. The cells were also stained with DAPI and visualized under fluorescence microscope. D: PRMT5 preferentially associates with unmethylated rRNA promoter in serum starved HeLa cells. Formaldehyde crosslinked chromatin prepared from serum starved (−FBS) and serum replenished (+FBS) HeLa cells was immunoprecipitated with anti-PRMT5 antibody (PRMT5) or preimmune sera (Preimn), and rRNA promoter was amplified from immunoprecipitated DNA and input DNA (Input). E: The immunoprecipitated DNA was either left undigested, or digested with Hpa II or Msp I. rRNA promoter was amplified from uncut, Hpa II and Msp I digested DNA by semi-quantitative PCR. The bar diagram represents PRMT5 binding to unmethylated and methylated rRNA promoter (as described in Fig. 2F).

Article Snippet: PRMT5 siRNA AND ITS TRANSFECTION HeLa cells were transfected at 70% confluency with 100 nM PRMTS siRNA (Santa Cruz, SC37022) or the control scrambled siRNA (Santa Cruz, SC37007) using Lipofectamine 2000.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Staining, Fluorescence, Microscopy, Immunoprecipitation, Amplification, Binding Assay, Methylation

Journal:

Article Title: Methylation of Histone H3 and H4 by PRMT5 Regulates Ribosomal RNA Gene Transcription

doi: 10.1002/jcb.22432

Figure Lengend Snippet: Ectopic expression of PRMT5 in HeLa cells inhibits the activity of both unmethylated and methylated rRNA promoter. A: Schematic representation of the rRNA promoter/luciferase reporter plasmid (pHrD-IRES-Luc) used in the transfection study. B: HeLa cells were transiently transfected with Flag tagged PRMT5 expression vector. The expression of PRMT5 protein was monitored by Western blot analysis using anti-Flag M2-monoclonal antibody. C: HeLa cells were transiently transfected with empty vector (0) or 2 and 4 µg of PRMT5 expression vector along with pHrD-IRES-Luc and the internal control pRL-TK. Cells harvested 48 h after transfection were analyzed for luciferase activity and normalized to pRL-TK. D: HeLa cells were transiently transfected with empty vector (0) and 4 µg of PRMT5 expression vector along with unmethylated or methylated pHrD-IRES-Luc and pRL-TK. Cells harvested 48 h after transfection were analyzed for luciferase activity and normalized to pRL-TK. E: HeLa cells were transfected with PRMT5 siRNA or control siRNA and expression of PRMT5 was monitored by western blot analysis at 24, 48, and 72 h after transfection. F: HeLa cells transfected with PRMT5 siRNA or control siRNA were harvested 24 h posttransfection and rRNA synthesis was monitored by real time PCR. Error bars represents standard deviation of triplicate measurements.

Article Snippet: PRMT5 siRNA AND ITS TRANSFECTION HeLa cells were transfected at 70% confluency with 100 nM PRMTS siRNA (Santa Cruz, SC37022) or the control scrambled siRNA (Santa Cruz, SC37007) using Lipofectamine 2000.

Techniques: Expressing, Activity Assay, Methylation, Luciferase, Plasmid Preparation, Transfection, Western Blot, Control, Real-time Polymerase Chain Reaction, Standard Deviation

Journal: PLoS ONE

Article Title: Hypomethylation and Genetic Instability in Monosomy Blastocysts May Contribute to Decreased Implantation Potential

doi: 10.1371/journal.pone.0159507

Figure Lengend Snippet: Genes involved in DNA methylation processes (including two housekeeping genes) and qPCR efficiency information (Taqman ® assays; Life Technologies).

Article Snippet: PRMT5 , 10419 / Hs01047356_m1 , -3.77 , 14 , Chromatin modifying protein.

Techniques: DNA Methylation Assay

Journal: PLoS ONE

Article Title: Hypomethylation and Genetic Instability in Monosomy Blastocysts May Contribute to Decreased Implantation Potential

doi: 10.1371/journal.pone.0159507

Figure Lengend Snippet: Ct values were normalized to RPL19 , an internal, constant housekeeping gene. Fold change was determined using the ΔΔCt method on the average of technical duplicates. Error bars represent standard error and the y-axis denotes fold change between euploid and aneuploidy. A) The expression of DNA methylatransferases was analyzed in euploid, trisomy 15, and monosomy 15 blastocysts (*P < 0.05). B) The expression of post-translational regulators and the chromatin modifying protein, PRMT5 (*P < 0.05).

Article Snippet: PRMT5 , 10419 / Hs01047356_m1 , -3.77 , 14 , Chromatin modifying protein.

Techniques: Expressing

Journal: Oncogene

Article Title: A TGFβ-PRMT5-MEP50 Axis Regulates Cancer Cell Invasion through Histone H3 and H4 Arginine Methylation Coupled Transcriptional Activation and Repression

doi: 10.1038/onc.2016.205

Figure Lengend Snippet: A. Dotplot of Wilcoxon rank-sum test p-values for PRMT5 and MEP50 expression in cancers with data collected from the TCGA. Upregulated expression (purple) and downregulated (green) are shown in dots scaled by–log( p ). Corresponding cancer cell lines probed in this study are bolded. B. Heatmap of gene expression of PRMT5 and MEP50 from TCGA lung cancer RNA-Seq data in normal and patients with lung adenocarcinoma (LUAD) or squamous cell carcinoma (LUSC), ranked by sample type (tumor or normal tissue) and then Shedden poor survival markers showing substantially increased expression of PRMT5 and MEP50 relative to normal tissue and poor survival markers. C. Dot-plot showing distribution of TCGA individual sample data for LUAD or LUSC; mean FPKM expression RNA-Seq value shown by black bar; Comparison between tumor and normal tissue expression p < 0.001 from Mann-Whitney Rank Sum Test. D. Whole cell lysate immunoblots for PRMTs and MEP50 in the normal and corresponding cancer cell lines. GAPDH is a loading control. E. Whole cell lysate and chromatin extracted from IMR90 and A549 probed with indicated antibodies. GAPDH and H3 are controls for lysate and chromatin immunoblots, respectively. F. Immunoblots for PRMT5 or MEP50 from A549 cells expressing shRNA targeted against GFP as a control (GFP kd ), against PRMT5 (PRMT5 kd ) or against MEP50 (MEP50 kd ). G. Representative immunofluorescence images for PRMT5 (red) or MEP50 (red) and counterstained with DAPI (blue) from GFP kd , PRMT5 kd , and MEP50 kd A549 cells demonstrating primarily cytoplasmic localization. Scale bar, 50 μm. H. Blue-native gel immunoblots for PRMT5 or MEP50 from knockdown A549 cells as in E. Native Molecular mass markers are indicated. Recombinant human PRMT5-MEP50 complex ( Hs PRMT5-MEP50) or SDS/boiled Hs PRMT5-MEP50 with SDS (lanes 1 and 2) are used for identification of intact complex (indicated) or free PRMT5 and MEP50, respectively.

Article Snippet: Purified human PRMT5 (Sinobiological) and PRMT5 in complex with MEP50 were prepared as described and used as controls.

Techniques: Expressing, RNA Sequencing Assay, MANN-WHITNEY, Western Blot, shRNA, Immunofluorescence, Recombinant

Journal: Oncogene

Article Title: A TGFβ-PRMT5-MEP50 Axis Regulates Cancer Cell Invasion through Histone H3 and H4 Arginine Methylation Coupled Transcriptional Activation and Repression

doi: 10.1038/onc.2016.205

Figure Lengend Snippet: A. FPKM ( F ragments p er k ilobase of transcript per m illion mapped reads) of all 9 PRMTs (CARM1 is PRMT4) and MEP50 from the 3 RNA-Seq replicates for each of the control (GFP kd ) and PRMT5 kd and MEP50 kd are shown on an arbitrary scale for each. PRMT5 and MEP50 reduced expression in each knockdown is indicated with the corresponding fold change from DESeq2 analysis. B. Top: piechart showing up- (purple) and down- (green) regulated genes as calculated by both DESeq2 and edgeR algorithms with cutoffs as indicated. Bottom: Venn diagram showing the overlap in the total geneset of altered expression between PRMT5 kd and MEP50 kd . C. Heatmap of the 955 genes altered in both knockdowns, showing PRMT5 and MEP50 up- (purple) and down- (green) regulated genes. The calculated Pearson correlation between the two knockdowns is shown. D. Selected ConsensusPathDB output genes in protein complexes are shown, colored by up- (purple) and down- (green) regulation. Bolded genes are viewed in E. E. IGV genome browser view of the RNA-Seq profile from a single replicate of the GFP kd , PRMT5 kd, and MEP50 kd analysis. The fibrinogen locus (chr4: FGB, FGA, and FGG) is shown as an example of downregulation upon knockdown (left panel, shaded green) and collagen 5α (chr9: COL5A1) is shown as an example of upregulation upon knockdown (right panel, shaded purple). F. Example gene set enrichment analysis of the 955 genes altered in both knockdowns. Top panel: adhesion molecules known to be downregulated in lung cancer had a significant positive normalized enrichment score (NES) upon knockdown. Bottom panel: a gene set known to be upregulated in lung and breast cancers had a significant negative NES upon knockdown. G. Dotplot of gene ontology terms enriched on both PRMT5 and MEP50 knockdown upregulated genes (top panel, purple) and downregulated genes (bottom panel, green) are shown in dots scaled by–log( p ). H. Dotplot of Ingenuity Pathway Analysis (IPA) diseases and functions enriched in both knockdowns are shown in dots scaled by–log( p ). I. Dotplot of Ingenuity Pathway Analysis (IPA) upstream pathways enriched in both knockdowns are shown in dots scaled by–log( p ).

Article Snippet: Purified human PRMT5 (Sinobiological) and PRMT5 in complex with MEP50 were prepared as described and used as controls.

Techniques: RNA Sequencing Assay, Expressing

Journal: Oncogene

Article Title: A TGFβ-PRMT5-MEP50 Axis Regulates Cancer Cell Invasion through Histone H3 and H4 Arginine Methylation Coupled Transcriptional Activation and Repression

doi: 10.1038/onc.2016.205

Figure Lengend Snippet: A. Proliferation of A549 cells expressing shRNA targeted against GFP (red circle; GFP kd ), PRMT5 (blue square; PRMT5 kd ) and MEP50 (orange triangle; MEP50 kd ). Plotted data are mean ± S.E.M. of three independent experiments. * p < 0.05 from one-way ANOVA test. Histogram (right): cell count on 6 th day for each sample. B. Colony formation assays of A549 cells expressing shRNAs are quantified for relative colony numbers (% of control). +Values are means ± S.E.M. of three independent experiments. * p < 0.05 from one-way ANOVA test. C. Migration through 8 μm pores by GFP kd , PRMT5 kd or MEP50 kd A549 cells was measured. Left: representative crystal violet staining of migrated cells on the underside of the porous polycarbonate membrane under a phase-contrast microscope (20X). Right: quantification of the migrated cells. Values are mean ± S.E.M. of three independent experiments. * p < 0.05 from one-way ANOVA test. D. Matrigel invasion through 8 μm pores by GFP kd , PRMT5 kd or MEP50 kd A549 cells was measured. Left: representative crystal violet staining of invaded cells on the underside of the porous polycarbonate membrane under a phase-contrast microscope (20X). Right: quantification of invaded cells. Values are mean ± S.E.M. of three independent experiments. * p < 0.05 from one-way ANOVA test. E. Rate of wound-healing of GFP kd , PRMT5 kd or MEP50 kd A549 cells was measured over 24 hours. Top: phase-contrast pictures (10X) of each cell line after scratching the confluent cells with the leading edge of cells indicated by a dashed yellow line. Bottom: quantification of the wound width (% 0hr wound width) after the scratch in indicated time points. Values are means ± S.E.M. of three independent experiments. * p < 0.05 from one-way ANOVA test. F. 3D spheroid cell invasion assay of GFP kd , PRMT5 kd or MEP50 kd A549 cells. Cells were aggregated into spheroids and then induced to invade the invasion matrix for the indicated time courses. The flat area of the cell mass view was calculated at four time points to measure cell invasion rate. Top: representative spheroid images for individual A549 cell lines under a phase-contrast microscope (20X). Bottom: histogram depicting the Spheroid area (×10 6 μm 2 ) measured with ImageJ 1.49. Values are means ± S.E.M. of three independent experiments. * p < 0.05 from one-way ANOVA test. G. A549 cells were treated with 0, 10nM, 100nM, 500nM, 1μM, or 5μM GS591 for 4 days and lysates or extracted histones were blotted for PRMT5, SYM10 (methylated SmD3), GAPDH (control), H4R3me2s, and H3 as indicated. DB71 stain of extracted histones is also shown. H. Invasivity of A549 cells treated with 500nM GSK591 for 0 or 4 days were measured in a Matrigel assay. Top: Representative crystal violet staining of invaded cells on the underside of the porous polycarbonate membrane through a phase-contrast microscope (20X) are shown. Bottom: Quantification of the invaded cells. Values are mean ± S.E.M. of three independent experiments. * p < 0.05 from one-way ANOVA test. I. Morphology (top micrographs) and viability (bottom plot) of A549 or IMR90 cells treated with 5 μM GSK591 for 4 days. Values are mean ± S.E.M. of three independent experiments. * p < 0.05 from one-way ANOVA test.

Article Snippet: Purified human PRMT5 (Sinobiological) and PRMT5 in complex with MEP50 were prepared as described and used as controls.

Techniques: Expressing, shRNA, Cell Counting, Migration, Staining, Microscopy, Invasion Assay, Methylation, Matrigel Assay

Journal: Oncogene

Article Title: A TGFβ-PRMT5-MEP50 Axis Regulates Cancer Cell Invasion through Histone H3 and H4 Arginine Methylation Coupled Transcriptional Activation and Repression

doi: 10.1038/onc.2016.205

Figure Lengend Snippet: A. Representative cell morphology images of GFP kd , PRMT5 kd or MEP50 kd A549 cells. Cells were treated with (+) or without (−) 10 nM TGFβ1 for 2 days. Inserts show higher magnification views of selected cells. B. Invasivity of GFP kd , PRMT5 kd or MEP50 kd A549 cells treated with (+) or without (−) 10 nM TGFβ1 for 2 days were measured in a Matrigel assay. Left: Representative crystal violet staining of invaded cells on the underside of the porous polycarbonate membrane through a phase-contrast microscope (20X) are shown. Right: Quantification of the invaded cells (+TGFβ, blue bars). Values are mean ± S.E.M. of three independent experiments. * p < 0.05 from one-way ANOVA test. C. Top: Images of a 3D spheroid cell invasion assay of GFP kd , PRMT5 kd or MEP50 kd A549 cells treated with (+) or without (−) 10 nM TGFβ1 for 2 days are shown. Bottom: Quantification of spheroid area (x10 6 μm 2 ) measured with ImageJ 1.49 (+TGFβ, blue bars). Values are means ± S.E.M. of three independent experiments. * p < 0.05 from one-way ANOVA test. D. Representative cell morphology images of A549 cells treated with (+) or without (−) 500 nM GSK591 for 4 days and (+) or without (−) 10 nM TGFβ1 for 2 days. Inserts show higher magnification views of selected cells. E. Invasivity of A549 lung adenocarcinoma cells, F. SK-MES-1 lung squamous cell carcinoma cells, and G. HS578T breast carcinoma cells treated with (+) or without (−) 500 nM GSK591 for 4 days and (+) or without (−) 10 nM TGFβ1 for 2 days measured in a Matrigel assay. Top: Representative crystal violet staining of invaded cells on the underside of the porous polycarbonate membrane through a phase-contrast microscope (20X) are shown. Bottom: Quantification of the invaded cells (+TGFβ, blue bars). Values are mean ± S.E.M. of three independent experiments. * p < 0.05 from one-way ANOVA test.

Article Snippet: Purified human PRMT5 (Sinobiological) and PRMT5 in complex with MEP50 were prepared as described and used as controls.

Techniques: Matrigel Assay, Staining, Microscopy, Invasion Assay

Journal: Oncogene

Article Title: A TGFβ-PRMT5-MEP50 Axis Regulates Cancer Cell Invasion through Histone H3 and H4 Arginine Methylation Coupled Transcriptional Activation and Repression

doi: 10.1038/onc.2016.205

Figure Lengend Snippet: A. Immunoblots for PRMTs, EMT markers, spliceosome factors, histone methylarginine PTMs, and controls as indicated, from A549 cells expressing shRNA targeted against GFP as a control (GFP kd ), against PRMT5 (PRMT5 kd ) or against MEP50 (MEP50 kd ). These cells were treated with (+) or without (−) 10 nM of TGFβ1 for 2 days. B. Immunoblots for the indicated proteins from A549 cells treated with (+) or without (−) 10 nM of TGFβ1 for 2 days and treated with (+) or without (−) 500 nM GSK591 for 4 days. C. Immunoblots for PRMT5, MEP50, and histone methylarginine PTMs from H460 lung cancer cells and D. HS578T breast cancer cells treated with (+) or without (−) 10 nM of TGFβ1 for 2 days and treated with (+) or without (−) 500 nM GSK591 for 4 days. E. qRT-PCR of relative mRNA expression of A549 cells treated with (+, blue bars) or without (−) 10 nM TGFβ1 for 2 days.

Article Snippet: Purified human PRMT5 (Sinobiological) and PRMT5 in complex with MEP50 were prepared as described and used as controls.

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

Journal: Oncogene

Article Title: A TGFβ-PRMT5-MEP50 Axis Regulates Cancer Cell Invasion through Histone H3 and H4 Arginine Methylation Coupled Transcriptional Activation and Repression

doi: 10.1038/onc.2016.205

Figure Lengend Snippet: A. Relative mRNA levels of indicated genes in A549 cells (control, PRMT5 kd and MEP50 kd , as indicated on top) treated with 10 nM of TGFβ1 (blue bars) for 2 days were determined by qRT-PCR. β-Actin was used as an internal control. Values are means ± S.E.M. of three independent experiments. * p < 0.05 from two-tailed one-way ANOVA test. B. qRT-PCR for the indicated genes from A549 cells expressing shRNA targeted against GFP as a control (GFP kd ), against PRMT5 (PRMT5 kd ) or against MEP50 (MEP50 kd ). These cells were treated with (+, blue bars) or without (−) 10 nM of TGFβ1 for 2 days as indicated. Values are means ± S.E.M. of three independent experiments. * p < 0.05 from one-way ANOVA test. C. Heatmap generated by ChIP-qPCR values to demonstrate the histone methylarginine code surrounding the candidate genes measured in (A). ChIP-qPCR enrichments from H3R2me1, H3R2me2s, H3R8me2s, H4R3me1, H4R3me2a, H4R3me2s and histone H3 from A549 on primers −1kb, at the promoter, or +1kb of the indicated genes are arrayed from blue (no enrichment) to yellow (maximal enrichment). ChIP-qPCR of TGFβ1-downregulated genes are arrayed on the top and TGFβ1-upregulated genes are on the bottom. D. Selected ChIP-qPCR experiments of alternatively up-regulated (Snail1 and Vim) and down-regulated genes (CDH1 and SPDEF) from A549 cells (control, PRMT5 kd and MEP50 kd , as indicated on bottom) treated with 10 nM of TGFβ1 (blue bars) for 2 days are shown. The horizontal dotted line indicates the upper limit of the 95% confidence interval of the signal from no-antibody (NA) control ChIPs. Error bars, SEM (n=3 independent cell passages). * p < 0.05 from one-way ANOVA test.

Article Snippet: Purified human PRMT5 (Sinobiological) and PRMT5 in complex with MEP50 were prepared as described and used as controls.

Techniques: Quantitative RT-PCR, Two Tailed Test, Expressing, shRNA, Generated

Journal: Oncogene

Article Title: A TGFβ-PRMT5-MEP50 Axis Regulates Cancer Cell Invasion through Histone H3 and H4 Arginine Methylation Coupled Transcriptional Activation and Repression

doi: 10.1038/onc.2016.205

Figure Lengend Snippet: A. Immunoblots for WDR5 and GAPDH from whole cell lysates of A549 treated with (+, blue bars) or without (−) TGFβ1 and with (+) or without (−) siRNA against WDR5. B. qRT-PCR of relative expression of Snail and Vimentin in A549 cells treated with (+, blue bars) or without (−) TGFβ1 and with (+) or without (−) OICR-9429, a small molecule inhibitor of the WDR5:H3 interaction. C. RT-qPCR as described in A except with A549 cells with (+, blue bars) or without (−) TGFβ1 and with (+) or without (−) expressing siRNA againstWDR5. D. H3R2me1 and H3K4me3 ChIP-qPCR experiments of up-regulated genes (Snail1 and Vim) in GFP kd and PRMT5 kd A549 cells treated with (+, blue bars) or without (−) TGFβ1. E. H3R2me1 and H3K4me3 ChIP-qPCR experiments of up-regulated genes (Snail1 and Vim) in A549 treated with (+, blue bars) or without (−) TGFβ1 and with (+) or without (−) OICR-9429. F. ChIP-qPCR as described in A except with A549 cells with (+, blue bars) or without (−) TGFβ1 and with (+) or without (−) expressing siRNA against WDR5. G. Model depicting the TGFβ1-PRMT5-MEP50 histone code axis involved in lung cancer metastasis progression. TGFβ1 stimulates PRMT5 protein expression and activity towards H4R3me2s (left, histone tail exaggerated for illustration), downregulating expression of cancer metastasis suppressor genes, and towards H3R2me1 (right), stimulating expression of EMT activator genes through recruitment of WDR5 and MLL to methylate H3K4me3. The targeted genes are central to regulation of cellular movement, EMT, and metastasis. Knockdown of PRMT5 or MEP50 can prevent this axis. A novel, specific, and highly potent PRMT5 inhibitor GSK591 also inhibits TGFβ1 induced cancer phenotypes with limited effect on survival of normal lung cells.

Article Snippet: Purified human PRMT5 (Sinobiological) and PRMT5 in complex with MEP50 were prepared as described and used as controls.

Techniques: Western Blot, Quantitative RT-PCR, Expressing, Activity Assay