Journal: Journal of ethnopharmacology

Article Title: Dehydrocorydaline attenuates myocardial ischemia-reperfusion injury via the FoXO signalling pathway: A multimodal study based on network pharmacology, molecular docking, and experimental study.

doi: 10.1016/j.jep.2024.118738

Figure Lengend Snippet: Fig. 5. Prediction and validation of DHC and its potential targets by molecular docking and IHC analysis. (A) Molecular docking of STAT3 and DHC. (B) Molecular docking of MDM2 and DHC. (C) Molecular docking of CDK2 and DHC. (D) Molecular docking of PLK1 and DHC. (E) Molecular docking of CCND1 and DHC. (F) Binding of DHC to CCND1 as determined through microscale thermophoresis (MST). (G) Binding of DHC to MDM2 as determined through MST. (H) Binding of DHC to CDK2 as determined through MST. (I) Relative protein levels of p-FOXO1A (n = 3). (J) Representative IHC images of p-FOXO1A, CCND1, and p-MDM2. (K) Relative protein levels of CCND1 (n = 3). (L) Relative protein levels of p-MDM2 (n = 3). *indicates a significant difference compared with the sham group, # indicates a significant difference compared with the MIRI group: *p < 0.05, **p < 0.01, #p < 0.05, ##p < 0.01. DHC: dehydrocorydaline, IHC: immunohistochemical, MIRI: myocardial ischemia-reperfusion injury.

Article Snippet: The microscale thermophoresis (MST) technique was performed to validate the interaction between CCND1 (#HY-P72119, MedChemExpress, Shanghai, China), CDK2 (#HY-P70014, MedChemExpress, Shanghai, China), MDM2 (#HY-P701593, MedChemExpress, Shanghai, China) and DHC(#HY-N0674, MedChemExpress, Shanghai, China).

Techniques: Biomarker Discovery, Binding Assay, Microscale Thermophoresis, Immunohistochemical staining

Journal: Journal of ethnopharmacology

Article Title: Dehydrocorydaline attenuates myocardial ischemia-reperfusion injury via the FoXO signalling pathway: A multimodal study based on network pharmacology, molecular docking, and experimental study.

doi: 10.1016/j.jep.2024.118738

Figure Lengend Snippet: Fig. 7. Effects of DHC on the expression of predicted targets in the in vitro model of H/R injury. (A) Representative fluorescence images of the TUNEL assay. Photographs were taken at × 400 magnification. (B) Percentage of TUNEL-positive cells in each group. (C) Western blot showing the protein expression of cleaved- caspase 3 and cleaved-caspase 8 in each group. (D) Western blot showing the protein expression of p-FOXO1A, FOXO1A, CCND1, p-MDM2, and MDM2 in each group. (E) Relative protein levels of cleaved-caspase 3 and cleaved-caspase 8 measured in western blots (n = 3). (F) Relative protein levels of p-FOXO1A, FOXO1A, and p- FOXO1A/FOXO1A measured in western blots (n = 3). (G) Relative protein levels of p-MDM2, MDM2, and p-MDM2/MDM2 measured in western blots (n = 3). (H) Relative protein levels of CCND1 measured in western blots (n = 3). * indicates a significant difference compared to the control group; # indicates a significant difference compared to the H/R model group: *p < 0.05, **p < 0.01, #p < 0.05, ##p < 0.01. DHC: dehydrocorydaline, H/R: hypoxia/reoxygenation, TUNEL: TdT- mediated dUTP-biotin nick end labelling.

Article Snippet: The microscale thermophoresis (MST) technique was performed to validate the interaction between CCND1 (#HY-P72119, MedChemExpress, Shanghai, China), CDK2 (#HY-P70014, MedChemExpress, Shanghai, China), MDM2 (#HY-P701593, MedChemExpress, Shanghai, China) and DHC(#HY-N0674, MedChemExpress, Shanghai, China).

Techniques: Expressing, In Vitro, Fluorescence, TUNEL Assay, Western Blot, Control

Journal: Nature Communications

Article Title: Concurrent BMP7 and FGF9 signalling governs AP-1 function to promote self-renewal of nephron progenitor cells

doi: 10.1038/ncomms10027

Figure Lengend Snippet: ( a , b ) CCNE1 (G1-phase) and PCNA (S-phase) immunostaining of E14.5 and E17.5 NPCs treated for 24 h with BMP7, TAK1 and JNK inhibitors. Graphs show the percentage of G1, G1-S and S-phase cells per condition. Three biological replicates analysed per condition ( n =3). Error bars represent s.d. ** P <0.005 and ** P <0.001, Student's t -test. ( c , d ) RT–qPCR of JUN and MYC targets in E14.5 and E17.5 NPCs treated with vehicle, BMP7, TAK1 and JNK inhibitors for 2 h. Error bars indicate s.d. Three biological replicates analysed per condition ( n =3). ( e , f ) RT–qPCR of JUN and MYC targets on NPCs isolated from E14.5 Jun het , Jun NP C and E17.5 Tak1 het , Tak1 NPC kidneys. Error bars represent s.d. Two biological replicates analysed per genotype ( n =2). ( g , h ) CCND1 and CCNE1 immunostaining in E14.5 Jun het , Jun NPC and E17.5 Tak1 C-WT , Tak1 C-NPC kidneys. Scale bars, 100 and 150 μM. Quantitation of CCND1+ and CCNE1+ cells per cap mesenchyme (yellow arrows) was calculated by scoring at least 30 random cap mesenchymes per kidney section per genotype for a total of 5 sections from each experimental group. Error bars indicate mean (s.d.). ** P <0.005 and ** P <0.001, Student's t -test. ( i ) Model for BMP7-TAK1-JNK-JUN pathway regulation of NPC self-renewal in early (E14.5) and later (E17.5) phases of nephrogenesis. CD, collecting duct; CM, cap mesenchyme.

Article Snippet: 3 × AP1-pGL3 was a gift from Dr Alexander Dent (Addgene # 40342) . pGL3Basic-962 CCND1 promoter luciferase), pGL3Basic-962 CCND1 promoter AP-1 site mutant (Addgene #32727 and # 32728) were gifts from Dr Frank McCormick . pRL-CMV (Renilla-Luciferase) was obtained from Promega.

Techniques: Immunostaining, Quantitative RT-PCR, Isolation, Quantitation Assay

Journal: Nature Communications

Article Title: Concurrent BMP7 and FGF9 signalling governs AP-1 function to promote self-renewal of nephron progenitor cells

doi: 10.1038/ncomms10027

Figure Lengend Snippet: ( a ) Immunostaining of EdU (S-phase) and pHH3 (M-phase), ( b ) CCNE1 (G1-phase) and PCNA (S-phase) in E17.5 NPCs stimulated with vehicle, BMP7, FGF9 or BMP7+FGF9 for 24 h. Scale bars, 50 μM. Graphs show the percentage of EdU+,pHH3+ cells and G1, G1-S and S-phase cells in each condition. Error bars represent mean (s.d.). ** P <0.005 and P <0.001 (Student's t -test). Two to three biological replicates analysed per condition ( n =2 in a ) and ( n =3 in b ) ( c , d ) RT–qPCR of cell cycle genes ( Ccnd1 and Myc ), Jun and Fos in NPCs stimulated with vehicle, BMP7, FGF9 or BMP7+FGF9 for 2 h. Error bars represent s.d. Three biological replicates analysed per condition, n =3. ( e , f ) pJUN and pFOS immunoblot of NPCs stimulated with vehicle, BMP7, FGF9 or BMP7+FGF9 for 20 min. Graph shows the relative density of pJUN and pFOS normalized to β-tubulin in each condition . ( g – i ) Bars in the graphs represent the average fold change in luciferase activity of 3xAP1-Luc, CCND1-Luc and CCND1 ΔAP-1 -Luc in NPCs stimulated with BMP7 and FGF9 relative to vehicle treatment for 24 h. Three biological replicates analysed per condition ( n =3). Error bars represent s.d. ** P <0.005, NS, not significant P >0.05, Student's t -test.

Article Snippet: 3 × AP1-pGL3 was a gift from Dr Alexander Dent (Addgene # 40342) . pGL3Basic-962 CCND1 promoter luciferase), pGL3Basic-962 CCND1 promoter AP-1 site mutant (Addgene #32727 and # 32728) were gifts from Dr Frank McCormick . pRL-CMV (Renilla-Luciferase) was obtained from Promega.

Techniques: Immunostaining, Quantitative RT-PCR, Western Blot, Luciferase, Activity Assay

Journal: Signal Transduction and Targeted Therapy

Article Title: E3 ligase MG53 suppresses tumor growth by degrading cyclin D1

doi: 10.1038/s41392-023-01458-9

Figure Lengend Snippet: MG53 induces G1 arrest via repressing cyclin D1. a Averaged data of flow cytometry showing MG53 induced cell cycle arrest in G1 phase in HCT116 ( n = 8). b Representative images of immunofluorescent signal of Ki67 in AGS cells with or without MG53 overexpression; n = 7. c HCT116 and AGS cell lysates were resolved on SDS-PAGE gel and stained with coomassie blue before mass spectrometry (MS) analysis. The red frame indicates the area on the SDS-PAGE gel that was excised for MS analysis. d Venn diagram showing the number of MG53-interacting proteins identified by MS. The blue circle represents the collection of proteins that interacted with MG53 in HCT116 cells, and the purple circle denotes the pool of proteins interacted with MG53 in AGS cells. The 409 proteins shared by these two collections were analyzed by KOBAS ( http://kobas.cbi.pku.edu.cn/ ) , and four cell cycle regulation-related proteins were identified (in red circle). e Overexpression of MG53 resulted in downregulation of D cyclins, but not other types of cyclins in AGS cells; n = 3. f Representative western blots showing the protein levels of MG53 and cyclin D1 in MG53-sensitive and -insensitive cell lines; n = 3. g , h Results of MTT assay showing the proliferation of human cancer cell lines in the presence of MG53 overexpression; n = 6. i Results of cell viability assay showing that overexpression of cyclin D1 attenuated cell death induced by MG53; n = 4. Scale bar = 50 μm in ( b ). Data were presented as mean ± s.e.m., and were analyzed using two-tailed unpaired t test. ns not significant, ** P < 0.01, and *** P < 0.001 as compared with the corresponding controls

Article Snippet: Cyclin D1 recombinant protein (#230-00261-10) was from Raybiotech.

Techniques: Flow Cytometry, Over Expression, SDS Page, Staining, Mass Spectrometry, Western Blot, MTT Assay, Viability Assay, Two Tailed Test

Journal: Signal Transduction and Targeted Therapy

Article Title: E3 ligase MG53 suppresses tumor growth by degrading cyclin D1

doi: 10.1038/s41392-023-01458-9

Figure Lengend Snippet: MG53 reduces cyclin D1 protein abundance and accelerates its turnover. a , b Results of western blots and RT-qPCR showing that overexpression of MG53 dose-dependently downregulated cyclin D1 at the protein levels without altering its mRNA levels in HCT116 ( a ) and AGS ( b ) cells; n ≥ 4. c , d Results of western blots and RT-qPCR showing cyclin D1 protein and mRNA levels in HCT116 ( c ) and SW480 ( d ) cells when MG53 expression was inhibited by specific shRNA; n = 6. e Representative western blots and statistical result showing that MG53 accelerated cyclin D1 protein turnover in HEK293 cells; n = 3. f , g Western blots showing that MG53-induced cyclin D1 degradation was blocked by proteasome inhibitor MG132 (10 μM, 12 h) in HCT116 ( f ) and AGS ( g ) cell lines; n = 6. Data are presented as mean ± s.e.m. and analyzed by two-tailed unpaired t test. ns, not significant, * P < 0.05, ** P < 0.01, and *** P < 0.001 as compared to the corresponding controls

Article Snippet: Cyclin D1 recombinant protein (#230-00261-10) was from Raybiotech.

Techniques: Quantitative Proteomics, Western Blot, Quantitative RT-PCR, Over Expression, Expressing, shRNA, Two Tailed Test

Journal: Signal Transduction and Targeted Therapy

Article Title: E3 ligase MG53 suppresses tumor growth by degrading cyclin D1

doi: 10.1038/s41392-023-01458-9

Figure Lengend Snippet: MG53 constitutes an E3 ligase targeting cyclin D1 for ubiquitination-dependent degradation. a Co-immunoprecipitation (co-IP) of endogenous MG53 with cyclin D1 in HCT116 cells. b Co-IP of MYC-tagged MG53 with endogenous cyclin D1 in AGS cells. c Co-IP of synthesized recombinant human MG53 (rhMG53) and cyclin D1 (rhCyclin D1) in vitro. d Ubiquitination of endogenous cyclin D1 was enhanced by overexpressing MG53 but not E3 ligase-inactive mutant MG53-C14A in HCT116 and AGS cells. e Ubiquitination of endogenous cyclin D1 was abated by silencing MG53 in HCT116 cells. f Representative western blots showing the K48-linked or K63-linked polyubiquitination of endogenous cyclin D1 in AGS cells. g Representative western blots of ubiquitination of exogenous cyclin D1 by MG53 with expressing wild type or mutant ubiquitin in HEK293 cells; WT, wild type ubiquitin; K 48 or K 63 , ubiquitin mutant containing only one lysine at position 48 or 63 while the rest of the lysine residues were mutated to arginine; K 48 R or K 63 R, ubiquitin mutant with lysine 48 or lysine 63 mutated to arginine. h Western blots showing MG53 mutants devoid of E3 ligase activity could not mediate the ubiquitination of cyclin D1 in HEK293 cells. i Representative western blots and averaged data showing MG53 mutants devoid of E3 ligase activity failed to repress the expression of cyclin D1; n = 6. j , k Representative western blots ( k ) showing polyubiquitination of FLAG-tagged wild type (WT) and various lysine mutants of cyclin D1 ( j ) in HEK293 cells with or without MG53 expression. l , m Western blots ( l ) and averaged data ( m ) showing MG53 failed to downregulate cyclin D1 K 33-238 R and K 33-239 R mutants; n = 4. a – h , k Three independent experiments were performed with similar results. Data are presented as mean ± s.e.m. and analyzed by two-tailed unpaired t test. ns, not significant, * P < 0.05 and ** P < 0.01 as compared to the corresponding controls

Article Snippet: Cyclin D1 recombinant protein (#230-00261-10) was from Raybiotech.

Techniques: Ubiquitin Proteomics, Immunoprecipitation, Co-Immunoprecipitation Assay, Synthesized, Recombinant, In Vitro, Mutagenesis, Western Blot, Expressing, Activity Assay, Two Tailed Test

Journal: Signal Transduction and Targeted Therapy

Article Title: E3 ligase MG53 suppresses tumor growth by degrading cyclin D1

doi: 10.1038/s41392-023-01458-9

Figure Lengend Snippet: Downregulation of MG53 is associated with increased cyclin D1 protein abundance in tumors. a , b Representative western blots and averaged data showing the protein levels of MG53 and cyclin D1 in colon tissues from MG53 transgenic (MG53-TG, a ) and MG53 knockout (MG53-KO, b ) mice compared to their corresponding wild type littermates (WT) after treatment with AOM/DSS; n ≥ 4. c , d Representative western blots ( c ) and averaged data ( d ) showing the expression of cyclin D1 in tumor tissues (T) compared to the corresponding adjacent normal tissues (N) from colorectal cancer patients; n = 46. e Regression analysis showing the protein level of MG53 is negatively correlated with that of cyclin D1 in tumor tissues from colorectal cancer patients; n = 46. f , g Representative western blots ( f ) and averaged data ( g ) showing the expression of cyclin D1 in tumor tissues (T) compared to the corresponding adjacent normal tissues (N) from gastric cancer patients; n = 11. h Regression analysis showing the protein level of MG53 is negatively correlated with that of cyclin D1 in tumor tissues from gastric cancer patients; n = 11. i Statistical results of tumor size and images of xenograft tumors in BALB/c nude mice derived from HCT116 cells expressing GFP or MG53-GFP treated with vehicle or palbociclib started when xenograft MG53-overexpressing tumors reached an average size of 100 mm 3 ; n ≥ 6. (Palb, palbociclib at 40 mg/kg administrated daily via oral gavage). j Schematic diagram showing E3 ligase MG53-mediated cyclin D1 degradation suppresses cancer growth. i GFP and MG53-GFP were HCT116-derived tumors expressing GFP and MG53-GFP, respectively using lentivirus infection. a , b , d , g , i Data were presented as mean ± s.e.m. and analyzed using two-tailed unpaired t test. * P < 0.05, ** P < 0.01, and *** P < 0.001 as compared with corresponding normal tissues

Article Snippet: Cyclin D1 recombinant protein (#230-00261-10) was from Raybiotech.

Techniques: Quantitative Proteomics, Western Blot, Transgenic Assay, Knock-Out, Expressing, Derivative Assay, Infection, Two Tailed Test

Journal: Nucleic Acids Research

Article Title: Novel RNA-binding activity of MYF5 enhances Ccnd1 / Cyclin D1 mRNA translation during myogenesis

doi: 10.1093/nar/gkw023

Figure Lengend Snippet: MYF5 target transcripts include mRNAs that encode proteins involved in myoblast proliferation and differentiation. ( A ) RIP assay using cytoplasmic lysates prepared from C2C12 cells using either anti-MYF5 antibody or IgG under conditions that preserved mRNA-RBP (mRNP) complexes. ( B ) Western blot analysis of MYF5 recovered in IP samples. ( C ) Following MYF5 RIP, MYF5-bound mRNAs were identified by microarray (RIP-chip) analysis in growing (GM) C2C12 cells. Data represent the Z -ratio of mRNAs in MYF5 RIP relative to IgG RIP. ( D ) RIP followed by RT-qPCR analysis to validate the association of MYF5 with mRNAs encoding myogenic proteins in proliferating C2C12 myoblasts; the levels of mRNAs in MYF5 IP were normalized to the levels of Gapdh mRNA and plotted as fold enrichment relative to the levels seen in control IgG IP samples. Discontinuous gray line: twofold enrichment in mRNAs bound to MYF5. ( E ) RIP analysis of Flag-MYF5 interaction with Ccnd1 mRNA. Forty-eight hours after C2C12 transfection with Flag-MYF5, RIP analysis was carried out using IgG or anti-Flag antibodies. Ccnd1 mRNA was detected by RT-qPCR analysis and its levels in Flag IP were compared with those in control IgG IP samples; Actn mRNA (encoding the housekeeping protein β-Actin) was measured to normalize sample input. Data in (D,E) represent the means and S.E.M. from three or more independent experiments. ( F ) Ingenuity pathway analysis (IPA) of mRNAs enriched in MYF5 IP relative to IgG in C2C12 growing myoblasts. *, P < 0.05 and **, P < 0.01 (Student's t -test).

Article Snippet: CCND1 siRNA (SR408533, Origene) was transfected 48 h before measuring cell number.

Techniques: Western Blot, Microarray, RNA Immunoprecipitation - Chromatin Immunoprecipitation, Quantitative RT-PCR, Control, Transfection

Journal: Nucleic Acids Research

Article Title: Novel RNA-binding activity of MYF5 enhances Ccnd1 / Cyclin D1 mRNA translation during myogenesis

doi: 10.1093/nar/gkw023

Figure Lengend Snippet: MYF5 binds to specific sequences on the Ccnd1 mRNA. (A) Top , schematic showing biotinylated RNA fragments spanning the 5′UTR, coding region (CR), and 3′UTR of Ccnd1 mRNA used for pulldown. Bottom , biotinylated RNA fragments were incubated with cytoplasmic lysates from C2C12 cells (GM); after pulldown using streptavidin beads, the levels of MYF5 bound to the biotinylated RNA segments were detected by western blot analysis. (B) Recombinant purified His-MYF5 was incubated with biotinylated Ccnd1 RNA fragments followed by pulldown and detection of MYF5 by western blot analysis using anti-MYF5 antibody. (C) Schematic of biotinylated RNA fragments spanning the Ccnd1 3′UTR-C transcript ( top ), were tested for binding to His-MYF5 after pull-down using streptavidin beads; His-MYF5 interaction with RNA segments of fragment C ( middle ) and smaller RNAs after closer subdivision of fragments 9 through 11 ( bottom ) were assessed by western blot analysis using anti-MYF5 antibody. (D) GST or GST-MYF5 were incubated with radiolabeled Ccnd1 3′UTR-C10–1, then either resolved on native acrylamide gels ( left ) by RNA electrophoretic mobility shift assay (EMSA), or crosslinked by UV irradiation and resolved by SDS-PAGE ( right ). (E) The domain of MYF5 that interacts with the Ccnd1 3′-C fragment was mapped by creating GST-tagged truncations of MYF5 ( left ) and testing their interaction by biotin pulldown and western blot analysis using anti-GST antibody ( right ).

Article Snippet: CCND1 siRNA (SR408533, Origene) was transfected 48 h before measuring cell number.

Techniques: Incubation, Western Blot, Recombinant, Purification, Binding Assay, Electrophoretic Mobility Shift Assay, Irradiation, SDS Page

Journal: Nucleic Acids Research

Article Title: Novel RNA-binding activity of MYF5 enhances Ccnd1 / Cyclin D1 mRNA translation during myogenesis

doi: 10.1093/nar/gkw023

Figure Lengend Snippet: MYF5 regulates CCND1 expression in C2C12 myoblasts. (A) Western blot analysis of MYF5 and CCND1 expression during C2C12 differentiation; heat shock protein 90 (HSP90) was included as loading control. (B,C) Forty-eight hours after transfecting C2C12 cells with MYF5 siRNA or Ctrl siRNA, the levels of MYF5, CCND1 and loading control GAPDH were analyzed by western blot analysis (B) and the levels of Ccnd1 pre-mRNA and mRNA by RT-qPCR analysis (C). (D–F) Forty-eight hours after transfection of proliferating C2C12 myoblasts with Ctrl or CCND1 siRNAs, the levels of CCND1 and HSP90 were assessed by western blot analysis (D) and cell numbers were measured using a TC10 automated cell counter (BioRad) and represented as fold change in cell number after CCND1 silencing relative to those in the Ctrl siRNA group (E). At day 6 in differentiation medium (DM6) C2C12 differentiation was monitored by measuring creatine kinase activity (F). (G) Forty-eight hours after MYF5 was overexpressed using pFlag-MYF5 pcDNA3, the levels of MYF5, CCND1 and loading control HSP90 were studied by western blot analysis. Data in (B-F) represent the means and S.E.M. from three or four independent experiments. *, P < 0.05 and **, P < 0.01 (Student's t -test).

Article Snippet: CCND1 siRNA (SR408533, Origene) was transfected 48 h before measuring cell number.

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

Journal: Nucleic Acids Research

Article Title: Novel RNA-binding activity of MYF5 enhances Ccnd1 / Cyclin D1 mRNA translation during myogenesis

doi: 10.1093/nar/gkw023

Figure Lengend Snippet: MYF5 promotes translation of Ccnd1 mRNA. (A,B) Forty-eight hours after Ctrl or MYF5 siRNA transfection of C2C12 cells, cytoplasmic extracts were fractionated through sucrose gradients to obtain cytoplasmic components of progressively larger weight: ribosomal subunits (40S, 60S), monosomes (80S) and low-molecular-weight (LMW) and high-molecular-weight (HMW) polysomes (A). The relative distribution of Gapdh mRNA, encoding a housekeeping protein, and Ccnd1 mRNA were measured by RT-qPCR analysis of RNA in each of the gradient fractions and represented as percentage of total RNA in the gradient (B). (C) Schematic of the dual luciferase reporter plasmids derived from the parent vector psiCHECK2 (psi), which expresses renilla luciferase (RL) and the internal control firefly luciferase (FL), and psiCHECK2-derived plasmids bearing the Ccnd1 fragments downstream of the RL coding region. (D) Top , Influence of MYF5 silencing on the expression of the reporter constructs. Twenty-four h after transfection of C2C12 cells with either MYF5 siRNA or Ctrl siRNA, each reporter plasmid was transfected, and 16 h later the ratio of RL activity to FL activity was measured. The decrease in relative RL/FL ratio of MYF5 siRNA-transfected cells relative to the RL/FL ratio of Ctrl siRNA-transfected cells is indicated. Bottom , RT-qPCR analysis of RL mRNA levels normalized to FL mRNA levels in each transfection group. Data represent the means and S.E.M. from 3 independent experiments. N.S., not significant; *, P < 0.05; **, P < 0.01 (Student's t -test).

Article Snippet: CCND1 siRNA (SR408533, Origene) was transfected 48 h before measuring cell number.

Techniques: Transfection, Molecular Weight, High Molecular Weight, Quantitative RT-PCR, Luciferase, Derivative Assay, Plasmid Preparation, Control, Expressing, Construct, Activity Assay

Journal: Nucleic Acids Research

Article Title: Novel RNA-binding activity of MYF5 enhances Ccnd1 / Cyclin D1 mRNA translation during myogenesis

doi: 10.1093/nar/gkw023

Figure Lengend Snippet: Influence of MYF5 on myogenesis via regulation of CCND1 expression. (A,B) C2C12 cells were transfected with MYF5 siRNA or Ctrl siRNA, along with a control vector [pcDNA3-Flag (pV)] or a plasmid vector that expressed Myc-tagged CCND1. Forty-eight hours later, the levels of CCND1 and MYF5 were analyzed by western blot analysis (A) and the degree of differentiation was analyzed by measuring creatine kinase activity at day 6 into differentiation (B). Data presented are the means and S.E.M. from four independent experiments; significance (P) is indicated. (C) Proposed model whereby MYF5 modulates myogenesis by acting upon CCND1 expression on two levels: first, MYF5 activates Ccnd1 transcription moderately, and second, MYF5 binds the Ccnd1 mRNA at the CR and 3′UTR, promoting Ccnd1 mRNA translation. The net effect is a CCND1-mediated increase in myoblast proliferation necessary at the initiation of myogenesis. N.S., not significant; *, P < 0.05; **, P < 0.01 (Student's t -test).

Article Snippet: CCND1 siRNA (SR408533, Origene) was transfected 48 h before measuring cell number.

Techniques: Expressing, Transfection, Control, Plasmid Preparation, Western Blot, Activity Assay