Journal: Cancer cell

Article Title: NF-?B-YY1-miR-29 Regulatory Circuitry in Skeletal Myogenesis and Rhabdomyosarcoma

doi: 10.1016/j.ccr.2008.10.006

Figure Lengend Snippet: (A) C2C12 cells were treated with TNFα and miR-29 was measured by qRT-PCR normalized to U6. Fold changes are shown with respect to vector cells where miR-29 levels were set to a value of 1. (B) MB were transfected with vector or a p65 plasmid and miR-29b/c levels were measured 48h post-transfection. (C) MB were transfected with either vector or p65 siRNA oligos and miR-29 expression was then measured as in (B). (D) MiR-29b/c were measured in MB stably expressing vector or IκBα-SR. Fold changes are shown with respect to vector cells, which were set to a value of 1. (E) MyoD was stably expressed in p65+/+ or p65−/− mouse embryonic fibroblasts (Bakkar et al., 2008) and qRT-PCR was performed for miR-29b and miR-29c. (F) ChIPs with YY1 or control IgG were performed on chromatins derived from either vector control (V) or Iκ Bα-SR (SR) expressing MB. Primers specific to site D were used for the PCR amplification. Total inputs are indicated.

Article Snippet: Source and dilutions for each antibody are as follows: YY1 (Santa Cruz Biotechnology; 1:1000), troponin T (Sigma; 1:1000), MyHC (Sigma; 1:1000), Ezh2 (Zymed; 1,000); p65 (Rockland; 1:10000), α-tubulin (Sigma; 1:1000), IκBα (Santa Cruz Biotechnology; 1:1000), and β-actin (Santa Cruz Biotechnology; 1:1000).

Techniques: Quantitative RT-PCR, Plasmid Preparation, Transfection, Expressing, Stable Transfection, Control, Derivative Assay, Amplification

Journal: Cancer cell

Article Title: NF-?B-YY1-miR-29 Regulatory Circuitry in Skeletal Myogenesis and Rhabdomyosarcoma

doi: 10.1016/j.ccr.2008.10.006

Figure Lengend Snippet: (A) C2C12 MB were induced to differentiate up to 4 days in differentiation medium (DM) and at indicated times miR-29a, miR-29b, and miR-29c were measured by qRT-PCR. (B) RT-PCR analysis of myogenic markers MyoD, myogenin, MyHC, skeletal actin (α-actin), troponin T performed at similar times to those in (A). (C) Westerns probing for YY1 and nuclear p65 in differentiating C2C12 cells. (D) Expression of miR-29b, and miR-29c in primary human myoblasts (GM) and myotubes (DM). (E) Measurement of miR-29 from either lower limb muscles at P2 and P8 or from TA muscles at P15, P23, and P90 in C57/BL6 mice. (F) Same measurement as in (E) from CTX injected TA muscles.

Article Snippet: Source and dilutions for each antibody are as follows: YY1 (Santa Cruz Biotechnology; 1:1000), troponin T (Sigma; 1:1000), MyHC (Sigma; 1:1000), Ezh2 (Zymed; 1,000); p65 (Rockland; 1:10000), α-tubulin (Sigma; 1:1000), IκBα (Santa Cruz Biotechnology; 1:1000), and β-actin (Santa Cruz Biotechnology; 1:1000).

Techniques: Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Expressing, Muscles, Injection

Journal: Cancer cell

Article Title: NF-?B-YY1-miR-29 Regulatory Circuitry in Skeletal Myogenesis and Rhabdomyosarcoma

doi: 10.1016/j.ccr.2008.10.006

Figure Lengend Snippet: (A) Extracts were prepared from normal human muscle and RMS cell lines and immunoblots were performed probing for YY1, Ezh2, and p65. (B) Lysates were prepared five RMS patient tumors and adjacent normal muscle tissue and probed for YY1, Ezh2, and p65 proteins. (C) RH30 cells were transfected with siRNA control oligos or siRNA-YY1. Cells were differentiated and semi-quantitative RT-PCR was performed probing for differentiation markers, or GAPDH used as a control. (D) RH30 tumors were established in nude mice and then injected with siRNA oligos every 3 days for 1 week. RNA and protein lysates were prepared from tumors and subsequently probed for MyHC, α-actin and troponin by RT-PCR and YY1 by Western. (E) ChIPs with either an YY1 antibody or control IgG were performed on chromatins isolated from human skeletal muscle cells (control) or RH30 cells. Precipitated DNA fragments were amplified with oligonucleotides spanning regions A-D of the human miR-29b/c regulatory region. Total inputs are indicated. (F) RH30 cells were infected with adenoviruses expressing vector control or IκBα-SR. YY1 and miR-29b levels were measured at 48h post-infection by qRT-PCR. (G) RH30 cells were infected with control or IκBá-SR adenovirus and differentiation markers were probed by qRT-PCR. (H) RH30 cells were transduced with vector or IκBα-SR expressing retroviruses to generate stable cell lines. Cells were then treated with differentiation medium for 2 days and immunostained for troponin or quantified for myofibrillar expression. (I) The model depicts the role of the NF-κB-YY1-miR-29 regulatory circuit in both normal myogenic differentiation and RMS. In myogenesis this circuit involves constitutive activity of NF-κB in myoblasts regulating YY1, which subsequently epigentically suppresses miR-29 and maintains cells in an undifferentiated state. As differentiation ensues, downregulation of the NF-κB–YY1 pathway leads to upregulation of miR-29 that in turns further decreases YY1 levels to ensure proper differentiation into myotubes. In RMS, this circuit becomes dysregulated due to an increase in the NF-κB–YY1 pathway that constitutively represses miR-29. In the absence of miR-29 tumor suppressor activity, YY1 is left uncontrolled thereby impairing differentiation leading to Rhabdomyosarcomagenesis.

Article Snippet: Source and dilutions for each antibody are as follows: YY1 (Santa Cruz Biotechnology; 1:1000), troponin T (Sigma; 1:1000), MyHC (Sigma; 1:1000), Ezh2 (Zymed; 1,000); p65 (Rockland; 1:10000), α-tubulin (Sigma; 1:1000), IκBα (Santa Cruz Biotechnology; 1:1000), and β-actin (Santa Cruz Biotechnology; 1:1000).

Techniques: Western Blot, Transfection, Control, Quantitative RT-PCR, Injection, Reverse Transcription Polymerase Chain Reaction, Isolation, Amplification, Infection, Expressing, Plasmid Preparation, Transduction, Stable Transfection, Activity Assay