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1.
Figure 1

Figure 1. From: twist is a potential oncogene that inhibits apoptosis.

Schematic outline of the screen designed to identify genes that confer protection from Myc-induced apoptosis.

Roberta Maestro, et al. Genes Dev. 1999 September 1;13(17):2207-2217.
2.
Figure 6

Figure 6. From: twist is a potential oncogene that inhibits apoptosis.

Twist and Dermo1 promote colony formation in soft agar. C8 MEFs were infected with retroviruses that direct the expression of LacZ, Twist, Dermo1, or Bcl-2. Infected cells were plated in soft agar, and colony formation was assessed after 2 weeks (as indicated). The LacZ-expressing cells form only a few, small colonies. Cells infected with Twist, Dermo1, or Bcl-2 form large colonies similar to those that are observed on expression of dominant-interfering alleles of p53. Expression of Twist or Dermo1 enhances colony formation by approximately five to sevenfold.

Roberta Maestro, et al. Genes Dev. 1999 September 1;13(17):2207-2217.
3.
Figure 7

Figure 7. From: twist is a potential oncogene that inhibits apoptosis.

Twist is highly expressed in rhabdomyosarcomas. (a) Formalin-fixed histologic sections of a rhabdomyosarcoma were stained with a Twist-specific antibody (SC-6070). Most of the neoplastic cells show nuclear accumulation of Twist protein. (b) Specificity of the signal was confirmed by loss of Twist immunoreactivity after preincubation of Twist antibody with the antigenic peptide. (c) A representative stain of a Twist-negative rhabdomyosarcoma is shown. (d) The lack of Twist immunoreactivity in differentiated skeletal muscle. Original magnification 400×.

Roberta Maestro, et al. Genes Dev. 1999 September 1;13(17):2207-2217.
4.
Figure 5

Figure 5. From: twist is a potential oncogene that inhibits apoptosis.

Twist down-regulates ARF. RNA was prepared from C8 cells infected with retroviruses that direct the expression of either Twist (T) or LacZ (G). ARF transcript was visualized by Northern blotting with an ARF-specific probe (exon 1β). For comparison, RNA was also prepared from BALBc 3T3 cells (B) that lack ARF expression and from primary MEFs (M) that are known to express ARF at high levels. The same blot was also probed for mouse β-actin as a control. Identical results are obtained from multiple independent infections and with cells plated under a variety of different conditions. Induction of apoptosis by treatment with adriamycin had no effect on the reduction of ARF mRNA by Twist.

Roberta Maestro, et al. Genes Dev. 1999 September 1;13(17):2207-2217.
5.

Figure 4. From: twist is a potential oncogene that inhibits apoptosis.

Twist interferes with the induction of p53-target genes. (A) p53 −/−MEFs were transfected with 1 μg of the p53-reporter plasmid (PG-13), a p53-expression plasmid (2 ng), and increasing amounts of Twist expression vector (pCDNA3–Twist). As an internal control, 0.5 μg of a LacZ-expressing plasmid was included in each transfection. At 24 hr post-transfection, cells were lysed and luciferase and β-galactosidase activities were measured (Promega). Values represent means ± s.e.m. of luciferase activities (normalized to β-galactosidase activities) from three independent transfections. (B) U2OS cells were transfected with 1 μg of the p53-reporter plasmid (PG-13), Twist-expressing vector (pCDNA3–Twist) as indicated, and 0.5 μg of LacZ plasmid. At 24-hr post-transfection, cells were processed and analyzed as described above. (C) C8 cells infected with either a Twist (T) or a control (lacZ, G) retrovirus were treated with adriamycin (adria, 0.2 μg/ml) for 10 hr. Total RNA was prepared from these and from parallel, untreated cultures (nt). Transcripts corresponding to p21, MDM2, and Bax were visualized by Northern blotting. For MDM2, only the major (3.3 kb) transcript is shown. The minor transcript (1.8 kb) showed the same relative response. All blots were probed with β-actin to control for RNA loading (a representative is shown).

Roberta Maestro, et al. Genes Dev. 1999 September 1;13(17):2207-2217.
6.

Figure 2. From: twist is a potential oncogene that inhibits apoptosis.

Twist protects from Myc-induced apoptosis. (A) Rat-1/MycER cells were infected with retroviral vectors (pHygroMarXII) that direct the expression of LacZ (a,d,g), Twist (b,e,h), or Bcl-2 (c,f,i), respectively. All cells were grown initially in 10% FBS. Cells were then exchanged into either 0.1% FBS plus 0.1 μm estradiol for 24 hr (d–f) or into 0.1% FBS for 8 days (g–i). (B) Protection from Myc-induced apoptosis was quantified by counting the number of apoptotic bodies (stained with Hoechst 33342) in cells that had been grown in 0.1% FBS plus 0.1 μm estradiol for 24 hr. Representative fields are shown. (C) For quantitation of apoptosis, cells that had been plated in 10% FBS were shifted into either 0.1% FBS plus 0.1 μm estradiol for 24 hr (left) or into 0.1% FBS for 72 hr (right). Twist- and Dermo1-infected populations are shown in comparison with lacZ-infected cells. The extent of apoptosis in each cell line is given with reference to the number of apoptotic bodies counted in the LacZ-infected population (100%). Bcl-2 and Mcl-1-infected cells were included as controls. Values represent means ± s.e.m. of three independent experiments. (D) Apoptosis of Rat-1/MycER cells following serum withdrawal was also quantified by counting viable cells. At the indicated times, adherent and nonadherent populations were collected and stained with trypan blue. Numbers of viable cells are given with reference to the starting populations. (●) Gal; (○) Twist; (▾) Dermo1. Values represent means ± s.d. of three independent experiments.

Roberta Maestro, et al. Genes Dev. 1999 September 1;13(17):2207-2217.
7.

Figure 3. From: twist is a potential oncogene that inhibits apoptosis.

Twist antagonizes p53. (A) E1A-Ras expressing MEFs (C8 MEFs) were infected with a retroviral vector (pBabe-puro) encoding LacZ, Twist, Dermo1, or Bcl-2, as indicated. Cells were plated in the presence of 10% FBS and after 24 hr were shifted to 0.1% FBS for 5 days. The result of a representative experiment is shown. Similar results were obtained after adriamycin-treatment (0.2 μg/ml) and after contact inhibition. (B) C8 MEFs infected with retroviruses that direct the expression of either Twist (shaded bars) or β-gal (solid bars) were treated with 0.2 μg/ml adriamycin for the indicated times. Viable cell numbers were determined by trypan blue staining and normalized to the starting cell number for each culture (100%). Values represent means ± s.d. for four independent experiments from two independent infections. (C) MEF-A3s were infected with retroviral vectors (pBabe-puro) that direct the expression of LacZ, Twist, or Bcl-2. After drug selection, cells were plated at low density and shifted to the permissive temperature of 32°C. Colony formation was monitored after 10 days. (D) C8 MEFs were infected with a control, LacZ (G) virus, or with viruses that direct the expression of Myc-tagged Twist (T) or Dermo1 (D). Protein expression was verified by Western blotting with a monoclonal antibody to the Myc tag (9E10).

Roberta Maestro, et al. Genes Dev. 1999 September 1;13(17):2207-2217.

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