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Results: 7

1.
Figure 5.

Figure 5. From: The Notch coactivator, MAML1, functions as a novel coactivator for MEF2C-mediated transcription and is required for normal myogenesis.

MAML1 and MEF2C act synergistically to activate a MEF2-dependent reporter. (A) MAML1 is not a coactivator for MyoD. C2C12 cells were transfected with 10 ng of Renilla luciferase plasmid, 0.5 μg of pMCK-luc reporter, and increasing amounts of expression plasmids encoding Flag-tagged MAML1 and/or MyoD. After 24 h transfection, cells were switched to DM, cultured overnight, and then lysed to prepare cell extracts for luciferase assay. The pMCK-luc reporter activities were expressed as fold activation relative to cells not expressing MAML1 and MyoD. (B) MAML1 activates a MEF2-responsive promoter in a dose-dependent manner. Assays were performed as described above, except the 3xMEF2-luc reporter is used. (C) MAML1 cooperates with MEF2C to activate MEF2-responsive promoter. Assays were performed as described above, except various amount of MEF2C expression vectors were cotransfected with MAML1.

Huangxuan Shen, et al. Genes Dev. 2006 March 15;20(6):675-688.
2.
Figure 4.

Figure 4. From: The Notch coactivator, MAML1, functions as a novel coactivator for MEF2C-mediated transcription and is required for normal myogenesis.

RNAi-mediated knockdown of Maml1 expression inhibits myogenesis in C2C12 cells. (A) RNAi-mediated Maml1 knockdown results in decreased expression of muscle proteins in C2C12 cells. C2C12 were transfected with two Maml1-specific siRNAs (V and VII), control siRNAs (Ctl), and water on two consecutive days, and cultured in DM at 48 h after the first transfection for 0, 2, or 4 d. Lysates were harvested for Western blotting. Note that MAML1 expression (shown at top, the upper band) is significantly (>80%–90%) decreased at day 0, but then returns to normal levels by day 2. However, myosin levels are significantly decreased in the cells, while myogenin and MyoD levels appear reduced as well. (B) Phase microscopy reveals that C2C12 cells with knocked down levels of MAML1 fail to form myotubes (panels 2,3), while cells transfected with control siRNAs or water (panels 1,4) form large, mature myotubes within 4 d of culture.

Huangxuan Shen, et al. Genes Dev. 2006 March 15;20(6):675-688.
3.
Figure 2.

Figure 2. From: The Notch coactivator, MAML1, functions as a novel coactivator for MEF2C-mediated transcription and is required for normal myogenesis.

Maml1 deficiency results in muscular dystrophy and defects in MyoD-induced myogenic conversion in MEFs that is rescued upon exogenous Maml1 expression. (A) Whole neonatal mice were sectioned and stained with H&E. (Panel 1) Hip muscles of a P6 heterozygous mouse shows normal fiber size and distribution. No differences were detected between heterozygotes and wild-type muscles (data not shown). (Panel 2) Hip muscles from a P6 Maml1 KO littermate have marked variation in muscle fiber size and the distribution of fibers is atypical. (Panel 3) Shoulder muscles of a P1 wild-type mouse show normal fiber size and distribution. (Panels 4,5) Advanced dystrophic phenotype in the shoulder muscles of a P1 Maml1 KO littermate showing many hypertrophic and atrophic muscle fibers, and, notably, sarcoplasmic vacuolization. The KO muscles also exhibit a noticeable increase in connective tissue. (B) Maml1 deficiency led to defects in MyoD-induced myogenic convesion in MEFs. Wild-type (right) and Maml1 KO (left) MEFs were infected with MyoD and cultured under differentiation medium for 3 d. (C) Wild-type (WT) MEFs have higher levels of myosin expression than the Maml1 KO MEFs. (− and +) Cells infected with retroviral vectors or viruses expressing MyoD. (D) Exogenous expression of MAML1 in KO MEFs rescues MyoD-induced myotube formation. Stably transduced MyoD KO MEF cells were infected with Flag-tagged MAML1 viruses or controls, and cells were induced for differentiation. (E) Expression of exogenous MAML1, MyoD, and Myosin were determined by Western blot analysis.

Huangxuan Shen, et al. Genes Dev. 2006 March 15;20(6):675-688.
4.
Figure 3.

Figure 3. From: The Notch coactivator, MAML1, functions as a novel coactivator for MEF2C-mediated transcription and is required for normal myogenesis.

Exogenous MAML1 expression enhances myogenic differentiation of C2C12 cells, and its N-terminal region is required for this effect. (A) Schematic diagram of FL and truncated MAML1 constructs in the pLXSN viral vectors used in this study. (B) Expression of exogenous MAML1 proteins in C2C12 cells. C2C12 cells were infected with pLXSN-based MAML1 viruses, and selected with G418. Expression of MAML1 proteins was confirmed by blotting with an anti-HA antibody. (C) Overexpression of MAML1 in C2C12 cells dramatically increases myotube formation and the N-terminal region of MAML1 is required for myogenic enhancement. Stably infected C2C12 Cells were induced to differentiate, and photographed at day 6. (D) Overexpression of MAML1 enhances myosin expression during myogenesis. Whole-cell lysates from infected C2C12 cells were prepared at days 0, 2, 4, and 6 after culturing in DM, and expression levels of muscle proteins were analyzed by Western blot analyses. (E) Expression of MAML1 enhances the activation of the promoter driving MCK. C2C12 cells were transfected with 10 ng of Renilla luciferase plasmid, 0.5 μg of pMCK-luc, and increasing amounts of expression plasmids encoding Flag-tagged MAML1. After 24 h transfection, cells were cultured in DM overnight and lysates were harvested. MCK reporter firefly luciferase activity, corrected for Renilla luciferase, is expressed as fold activation relative to cells not expressing MAML1.

Huangxuan Shen, et al. Genes Dev. 2006 March 15;20(6):675-688.
5.
Figure 6.

Figure 6. From: The Notch coactivator, MAML1, functions as a novel coactivator for MEF2C-mediated transcription and is required for normal myogenesis.

MEF2C and MAML1 interact in vivo. (A) MEF2C and MAML1 colocalize in the nucleus. U20S cells were transfected with the GFP-tagged MEF2C alone (top) or with Flag-tagged MAML1 (bottom), and stained with an anti-Flag antibody to detect MAML1 expression. (BG) Background staining. DAPI staining was performed to label the nuclei. Upon coexpression, MEF2C changed its localization from a diffuse nuclear pattern to colocalize with MAML1 in nuclear dots (merged image at bottom). (B) Coexpression of MAML1 and MEF2C results in post-translational modification of MEF2C. C2C12 cells were transfected with Flag-tagged MEF2C, with or without cotransfection of Flag-tagged MAML1. The expression of both MAML1 and MEF2C were detected by Western blot analysis with anti-Flag antibodies. Note that when the two proteins are coexpressed, the mobility of the MEF2C band shifts. (C) MAML1 interacts with the 87–177-amino-acid region of MEF2C by mammalian two-hybrid assays. C2C12 cells were cotransfected with a firefly luciferase reporter containing GAL4-binding sites (pSG5-luc) along with two expression vectors; i.e., one encoding DB or DB fused to FL or truncated MEF2C, and the other encoding AD or AD fused to MAML1. A total of 0.5 μg of each construct was used for transfection. Cellular lysates were harvested 44 h post-transfection. Firefly luciferase activity, normalized with Renilla luciferase expressed from the DB vector, was expressed as fold activation relative to the background level of firefly luciferase expression in the presence of empty DB and AD vectors. (D) MEF2C interacts with the N-terminal region of the MAML1, 1–70 amino acids by mammalian two-hybrid assays. Similar assays were carried out as described. FL MEF2C was expressed as DB fusion, while FL or truncated MAML1 expressed as AD fusion.

Huangxuan Shen, et al. Genes Dev. 2006 March 15;20(6):675-688.
6.
Figure 7.

Figure 7. From: The Notch coactivator, MAML1, functions as a novel coactivator for MEF2C-mediated transcription and is required for normal myogenesis.

Activation of the Notch signaling pathway overrides MAML1-enhanced myogenesis. (A) Notch ligand stimulation abrogated MAML1-mediated enhancement of myotube formation. C2C12 cells stably transduced with pLXSN based MAML1 viruses or vector control viruses were plated on culture wells coated with either control IgG or ligand Delta-Extra–IgG, and cultured in DM for 6 d. (B) Expression of a constitutively activated form of Notch1 (ICN1) inhibited MAML1-induced activation of MCK promoter. MAML1-transduced C2C12 cells or controls were transfected with 0.5 μg of pMCK-luc, 10 ng of Renilla luciferase plasmid, and various amounts of Notch ICN1, and luciferase assays were performed at 44–48 h after transfection. (C) Notch ICN1–MAML1–CSL complex formed in C2C12 cells. C2C12 cells were cotransfected with different combinations of expression plasmids encoding Flag-tagged MAML1, HA-tagged ICN1, Myc-tagged CSL, and Flag-tagged MEF2C as indicated. Whole-cell lysates (WCL) or anti-HA immunoprecipitates (IP) were blotted with anti-Flag or anti-HA, or anti-Myc antibodies. (D) Expression of ICN1 interfered with the coregulatory function of Maml1 on MEF2C-mediated transcription. C2C12 cells were transfected with 0.5 μg of a firefly luciferase reporter containing GAL4-binding sites (pSG5-luc), 0.5 μg of the plasmid encoding DB fused to MEF2C, and 0.5 μg of expression construct expressing Flag-tagged MAML1 in the presence of various amounts of HA-tagged ICN1 expression constructs (0, 0.25, and 0.5 μg). pSG5-luc firefly luciferase activity, corrected for Renilla luciferase activity, is expressed as fold activation relative to cells not expressing MAML1 and ICN1.

Huangxuan Shen, et al. Genes Dev. 2006 March 15;20(6):675-688.
7.
Figure 1.

Figure 1. From: The Notch coactivator, MAML1, functions as a novel coactivator for MEF2C-mediated transcription and is required for normal myogenesis.

Generation of Maml1-null mice reveals that it is required for normal development. (A) Diagram of the Maml1 targeting construct. Black boxes denote exons of the mouse Maml1 gene. Exon 1 was replaced with a neo gene in the indicated targeting vector. When genomic DNA is digested with XmnI, it is predicted that, using the denoted 5′-flanking probe, 11.7- and 23.3-kb bands would be detected for the wild-type (WT) and KO alleles, respectively. (B) Southern blotting indicated successful targeting of the Maml1 gene. Genomic DNA was isolated from embryos, digested with XmnI, blotted, and hybridized with the 5′-flanking probe. The predicted 23.3-kb band was detected in both the heterozygous and homozygous KO progeny, while the wild type and hetertozygotes contained the 11.7-kb fragment, as expected. (C) Northern blotting showed a loss of Maml1 transcripts in embryonic fibroblasts derived from a Maml1 KO embryo. Note that heterozygotes have reduced levels of Maml1 transcripts compared with wild-type transcripts. The size of the Maml1 transcript is ∼6 kb. Relative loading is indicated by 28S rRNA. (D) Western blotting confirmed a loss of MAML1 protein in embryonic fibroblasts derived from a Maml1 KO embryo and a reduction of the protein in the heterozygotes. MAML1 expression (∼140 kDa) was analyzed by immunoprecipitation with anti-MAML1 antibodies, followed by Western blotting. (E) Maml1 KO neonate (right) is growth retarded, as compared with its wild-type littermates (left), and die soon after birth (within 10 d). Mice are shown at P5. (F) A representative growth curve reveals that Maml1 KO neonates (black diamonds) fail to thrive, compared with wild-type (WT) littermates (gray squares). Data is presented as weight in grams at successive days after birth.

Huangxuan Shen, et al. Genes Dev. 2006 March 15;20(6):675-688.

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