pcmv myod1  (Addgene inc)

Journal: BMC Biology

Article Title: TIS7 induces transcriptional cascade of methylosome components required for muscle differentiation

doi: 10.1186/s12915-016-0318-6

Figure Lengend Snippet: TIS7 epigenetically regulates MyoD expression. a TIS7 KO MSCs were transfected with indicated plasmids. Quantitative PCR analysis of myoD was normalized to GAPDH expression (n = 3 independent experiments). Values represent the mean ± SEM and resulting data were analyzed via two-tailed type 2 Student’s t-test, *** P < 0.001 and * P < 0.05. b Proliferating TIS7 KO MSCs were transfected with indicated plasmids and total cell lysates were probed with MyoD and control antibodies. Quantification (n = 3 biological replicates) was performed by normalization to the tubulin signal. Values represent the mean ± SD and resulting data were analyzed via two-tailed type 2 Student’s t-test, *** P < 0.001 and ** P < 0.01. Representative western blots are shown. ChIP analyses using chromatin from TIS7 WT or KO MSCs transfected with either GFP or GFP-TIS7 plasmid DNA. c Schematic outline of mouse myoD gene locus. Primer pairs used for ChIP assays are indicated by arrows. The alignment of human and mouse sequences amplified by the PCR is shown. Antisera directed against symmetrically di-methylated histone H3 at arginine 8 ( d ) or PRMT5 ( e ) were used for immunoprecipitation. Immunoprecipitated DNA was analyzed by qPCR in triplicates using primers specific for the mouse myoD regulatory region. ChIP with control rabbit IgG or 1.23 % of total chromatin (input) were used as controls. Signals were normalized to input chromatin and shown as percentage of input, ** P ≤ 0.001 and *** P < 0.00005

Article Snippet: Full length mouse TIS7 (mTIS7), human PRMT5 (hPRMT5, Origen, cat. no. ORIGSC116333), mouse myoD (CMV-myc-tagged myoD; Addgene plasmid 8399; principal investigator: Andrew Lassar), and human ICln (hICln) cDNA or plasmid DNA were PCR amplified and cloned in frame with the fluorescent proteins ECFP and EYFP into the pECFP-C1, pECFP-N1, pEYFP-C1, or pEYFP-N1 (Clontech) mammalian expression vectors.

Techniques: Expressing, Transfection, Real-time Polymerase Chain Reaction, Two Tailed Test, Western Blot, Plasmid Preparation, Amplification, Methylation, Immunoprecipitation

Journal: BMC Biology

Article Title: TIS7 induces transcriptional cascade of methylosome components required for muscle differentiation

doi: 10.1186/s12915-016-0318-6

Figure Lengend Snippet: ICln affects in vivo methylation of histone H3 and binding of PRMT5 to myoD locus, resulting in increased myoD RNA and protein levels. ChIP analysis using chromatin from TIS7 WT or KO MSCs transfected with either YFP or YFP-ICln plasmids. Antisera directed against symmetrically dimethylated histone H3 at arginine 8 ( a ) or PRMT5 ( b ) were used for immunoprecipitation. Immunoprecipitated DNA was analyzed in triplicate by qPCR using primers to amplify the mouse myoD regulatory region. ChIP with control rabbit IgG or 1.23 % of total chromatin (input) were used as controls. Signals were normalized to input chromatin and shown as percentage of input. ** P ≤ 0.001 and *** P < 0.00005. c TIS7 KO MSCs were transfected with indicated plasmids and total cell lysates were probed with anti-MyoD and anti-YFP antibodies. Quantification (n = 3 biological replicates) was performed by normalization to the tubulin signal. Representative immunoblots are shown. d TIS7 KO MSCs were transfected with the indicated plasmids. Quantitative PCR analysis of myoD was normalized to GAPDH expression (n = 3 biological replicates and ** P < 0.01). e PRMT5 knockdown significantly increased myoD RNA levels in TIS7 KO MSCs transduced with sh PRMT5-expressing lentivirus. myoD RNA levels were measured by qPCR and PRMT5 protein levels were documented by immunoblots with indicated antibodies

Article Snippet: Full length mouse TIS7 (mTIS7), human PRMT5 (hPRMT5, Origen, cat. no. ORIGSC116333), mouse myoD (CMV-myc-tagged myoD; Addgene plasmid 8399; principal investigator: Andrew Lassar), and human ICln (hICln) cDNA or plasmid DNA were PCR amplified and cloned in frame with the fluorescent proteins ECFP and EYFP into the pECFP-C1, pECFP-N1, pEYFP-C1, or pEYFP-N1 (Clontech) mammalian expression vectors.

Techniques: In Vivo, Methylation, Binding Assay, Transfection, Immunoprecipitation, Western Blot, Real-time Polymerase Chain Reaction, Expressing, Transduction

Journal: BMC Biology

Article Title: TIS7 induces transcriptional cascade of methylosome components required for muscle differentiation

doi: 10.1186/s12915-016-0318-6

Figure Lengend Snippet: Model of myogenesis regulation by the TIS7/ICln/PRMT5 pathway. In skeletal muscles, TIS7 transcriptionally regulates ICln. The accumulated ICln interacts with PRMT5 and prevents its recruitment to chromatin-bound complexes on the myoD gene. This regulates PRMT5-mediated symmetrical dimethylation of histone H3 Arg8 and myoD expression. The regulated myoD level affects the balance between proliferation and differentiation of MSCs

Article Snippet: Full length mouse TIS7 (mTIS7), human PRMT5 (hPRMT5, Origen, cat. no. ORIGSC116333), mouse myoD (CMV-myc-tagged myoD; Addgene plasmid 8399; principal investigator: Andrew Lassar), and human ICln (hICln) cDNA or plasmid DNA were PCR amplified and cloned in frame with the fluorescent proteins ECFP and EYFP into the pECFP-C1, pECFP-N1, pEYFP-C1, or pEYFP-N1 (Clontech) mammalian expression vectors.

Techniques: Expressing

Journal: BMC Biology

Article Title: TIS7 induces transcriptional cascade of methylosome components required for muscle differentiation

doi: 10.1186/s12915-016-0318-6

Figure Lengend Snippet: TIS7 epigenetically regulates MyoD expression. a TIS7 KO MSCs were transfected with indicated plasmids. Quantitative PCR analysis of myoD was normalized to GAPDH expression (n = 3 independent experiments). Values represent the mean ± SEM and resulting data were analyzed via two-tailed type 2 Student’s t-test, *** P < 0.001 and * P < 0.05. b Proliferating TIS7 KO MSCs were transfected with indicated plasmids and total cell lysates were probed with MyoD and control antibodies. Quantification (n = 3 biological replicates) was performed by normalization to the tubulin signal. Values represent the mean ± SD and resulting data were analyzed via two-tailed type 2 Student’s t-test, *** P < 0.001 and ** P < 0.01. Representative western blots are shown. ChIP analyses using chromatin from TIS7 WT or KO MSCs transfected with either GFP or GFP-TIS7 plasmid DNA. c Schematic outline of mouse myoD gene locus. Primer pairs used for ChIP assays are indicated by arrows. The alignment of human and mouse sequences amplified by the PCR is shown. Antisera directed against symmetrically di-methylated histone H3 at arginine 8 ( d ) or PRMT5 ( e ) were used for immunoprecipitation. Immunoprecipitated DNA was analyzed by qPCR in triplicates using primers specific for the mouse myoD regulatory region. ChIP with control rabbit IgG or 1.23 % of total chromatin (input) were used as controls. Signals were normalized to input chromatin and shown as percentage of input, ** P ≤ 0.001 and *** P < 0.00005

Article Snippet: Full length mouse TIS7 (mTIS7), human PRMT5 (hPRMT5, Origen, cat. no. ORIGSC116333), mouse myoD (CMV-myc-tagged myoD; Addgene plasmid 8399; principal investigator: Andrew Lassar), and human ICln (hICln) cDNA or plasmid DNA were PCR amplified and cloned in frame with the fluorescent proteins ECFP and EYFP into the pECFP-C1, pECFP-N1, pEYFP-C1, or pEYFP-N1 (Clontech) mammalian expression vectors.

Techniques: Expressing, Transfection, Real-time Polymerase Chain Reaction, Two Tailed Test, Control, Western Blot, Plasmid Preparation, Amplification, Methylation, Immunoprecipitation

Journal: BMC Biology

Article Title: TIS7 induces transcriptional cascade of methylosome components required for muscle differentiation

doi: 10.1186/s12915-016-0318-6

Figure Lengend Snippet: ICln affects in vivo methylation of histone H3 and binding of PRMT5 to myoD locus, resulting in increased myoD RNA and protein levels. ChIP analysis using chromatin from TIS7 WT or KO MSCs transfected with either YFP or YFP-ICln plasmids. Antisera directed against symmetrically dimethylated histone H3 at arginine 8 ( a ) or PRMT5 ( b ) were used for immunoprecipitation. Immunoprecipitated DNA was analyzed in triplicate by qPCR using primers to amplify the mouse myoD regulatory region. ChIP with control rabbit IgG or 1.23 % of total chromatin (input) were used as controls. Signals were normalized to input chromatin and shown as percentage of input. ** P ≤ 0.001 and *** P < 0.00005. c TIS7 KO MSCs were transfected with indicated plasmids and total cell lysates were probed with anti-MyoD and anti-YFP antibodies. Quantification (n = 3 biological replicates) was performed by normalization to the tubulin signal. Representative immunoblots are shown. d TIS7 KO MSCs were transfected with the indicated plasmids. Quantitative PCR analysis of myoD was normalized to GAPDH expression (n = 3 biological replicates and ** P < 0.01). e PRMT5 knockdown significantly increased myoD RNA levels in TIS7 KO MSCs transduced with sh PRMT5-expressing lentivirus. myoD RNA levels were measured by qPCR and PRMT5 protein levels were documented by immunoblots with indicated antibodies

Article Snippet: Full length mouse TIS7 (mTIS7), human PRMT5 (hPRMT5, Origen, cat. no. ORIGSC116333), mouse myoD (CMV-myc-tagged myoD; Addgene plasmid 8399; principal investigator: Andrew Lassar), and human ICln (hICln) cDNA or plasmid DNA were PCR amplified and cloned in frame with the fluorescent proteins ECFP and EYFP into the pECFP-C1, pECFP-N1, pEYFP-C1, or pEYFP-N1 (Clontech) mammalian expression vectors.

Techniques: In Vivo, Methylation, Binding Assay, Transfection, Immunoprecipitation, Control, Western Blot, Real-time Polymerase Chain Reaction, Expressing, Knockdown, Transduction

Journal: BMC Biology

Article Title: TIS7 induces transcriptional cascade of methylosome components required for muscle differentiation

doi: 10.1186/s12915-016-0318-6

Figure Lengend Snippet: MSC differentiation is regulated by MyoD, ICln, and TIS7. a TIS7 WT and KO MSCs were plated at the same cell density. Proliferating TIS7 KO MSCs were transfected with indicated plasmids and 6 h later the differentiation was induced. After 5 d of differentiation, cells were fixed, stained with anti-myosin heavy chain antibody MF 20, and the nuclei were visualized using Hoechst. Scale bar = 100 μm. b Percentage of MSC fusion was calculated as the number of nuclei localized in multinucleated (n > 2) myotubes defined by MF20 staining compared to the total number of nuclei. Vertical bars denote the mean ± SD and were calculated from independent biological replicates (n = 3). The indicated number of nuclei was evaluated. Resulting data were analyzed via two-tailed type 2 Student’s t-test, *** P < 0.001. c Quantitative analysis of cells with the indicated nuclei numbers per cell defined by myosin staining. This analysis showed a significant decrease in the number of multinucleated cells and a completely missing category of cells with more than 15 nuclei in TIS7 KO MSCs when compared to TIS7 WT MSCs. Ectopic expression of TIS7, MyoD, or ICln significantly increased the number of multinucleated cells, mainly those with more than 15 nuclei in TIS7 KO MSCs when compared to TIS7 WT MSCs

Article Snippet: Full length mouse TIS7 (mTIS7), human PRMT5 (hPRMT5, Origen, cat. no. ORIGSC116333), mouse myoD (CMV-myc-tagged myoD; Addgene plasmid 8399; principal investigator: Andrew Lassar), and human ICln (hICln) cDNA or plasmid DNA were PCR amplified and cloned in frame with the fluorescent proteins ECFP and EYFP into the pECFP-C1, pECFP-N1, pEYFP-C1, or pEYFP-N1 (Clontech) mammalian expression vectors.

Techniques: Transfection, Staining, Two Tailed Test, Expressing

Journal: BMC Biology

Article Title: TIS7 induces transcriptional cascade of methylosome components required for muscle differentiation

doi: 10.1186/s12915-016-0318-6

Figure Lengend Snippet: Model of myogenesis regulation by the TIS7/ICln/PRMT5 pathway. In skeletal muscles, TIS7 transcriptionally regulates ICln. The accumulated ICln interacts with PRMT5 and prevents its recruitment to chromatin-bound complexes on the myoD gene. This regulates PRMT5-mediated symmetrical dimethylation of histone H3 Arg8 and myoD expression. The regulated myoD level affects the balance between proliferation and differentiation of MSCs

Article Snippet: Full length mouse TIS7 (mTIS7), human PRMT5 (hPRMT5, Origen, cat. no. ORIGSC116333), mouse myoD (CMV-myc-tagged myoD; Addgene plasmid 8399; principal investigator: Andrew Lassar), and human ICln (hICln) cDNA or plasmid DNA were PCR amplified and cloned in frame with the fluorescent proteins ECFP and EYFP into the pECFP-C1, pECFP-N1, pEYFP-C1, or pEYFP-N1 (Clontech) mammalian expression vectors.

Techniques: Muscles, Expressing