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Results: 7

1.
Figure 6.

Figure 6. From: Kinase-activating and kinase-impaired cardio-facio-cutaneous syndrome alleles have activity during zebrafish development and are sensitive to small molecule inhibitors.

Treatment of BRAF, MEK1 and MEK2 variants with SU-5402. (A) The mutant phenotypes promoted by the RNA expression of BRAF variants are prevented by pharmacological treatment with the FGFR1 inhibitor SU-5402, with the exception of the developmental phenotype caused by BRAFV600E. (B) Similarly, the elongation promoted by MEK1 and MEK2 disease variants is prevented by SU-5402 treatment. (C) Western blotting of total zebrafish lysates for ERK and phospho-ERK protein shows that SU-5402 treatment causes reduction of ERK phosphorylation, with α-tubulin as a loading control.

Corina Anastasaki, et al. Hum Mol Genet. 2009 July 15;18(14):2543-2554.
2.
Figure 3.

Figure 3. From: Kinase-activating and kinase-impaired cardio-facio-cutaneous syndrome alleles have activity during zebrafish development and are sensitive to small molecule inhibitors.

BRAF and MEK disease alleles promote cell movement phenotypes. (A) The most common CFC varient, BRAFQ257R, was expressed in developing embryos, and the embryos imaged every hour. Development of the embryos begins to become affected by BRAFQ257R expression between 7.5 and 8.5 hpf. (B) BRAF and (C) MEK disease variants promote significant changes in cell movement, as revealed by in situ hybridization. The gene expression domain of dlx3 is altered for both expression pattern and intensity of signal, such that there is no dlx3 detected in the BRAFV600E expressing embryos. HggI expression remains unaffected. The bottom panel shows the lateral view of the same embryos.

Corina Anastasaki, et al. Hum Mol Genet. 2009 July 15;18(14):2543-2554.
3.
Figure 4.

Figure 4. From: Kinase-activating and kinase-impaired cardio-facio-cutaneous syndrome alleles have activity during zebrafish development and are sensitive to small molecule inhibitors.

BRAF kinase-active and kinase-impaired alleles can promote an additive effect during development. Embryos co-expressing combinations of suboptimal doses (15 pg) of kinase-active (BRAFQ257R, BRAFS467A) and kinase-impaired (BRAFG596V) CFC alleles or BRAFWT mRNA were assessed for the elongation phenotype at 10 hpf. The number of elongated embryos did not change significantly upon expression of a single BRAF CFC allele (15 pg), or in combination with BRAFWT (for a total of 30 pg). A significant increase in the mutant phenotype was induced by co-injections of BRAFQ257R with BRAFS467A (P < 0.0001) or BRAFQ257R with BRAFG596V (P = 0.0003) compared with the BRAF CFC allele co-injected with BRAFWT as indicated by χ2 tests. The numbers in the bars indicates the percentages of elongated embryos; n is the number of injected embryos.

Corina Anastasaki, et al. Hum Mol Genet. 2009 July 15;18(14):2543-2554.
4.
Figure 1.

Figure 1. From: Kinase-activating and kinase-impaired cardio-facio-cutaneous syndrome alleles have activity during zebrafish development and are sensitive to small molecule inhibitors.

CFC syndrome alleles promote developmental changes during early embryogeneis. (A) RNA expression of CFC and melanoma variants BRAFQ257R (kinase-activating, CFC), BRAFG596V (kinase-impaired, CFC and melanoma) and BRAFV600E (very high-kinase, melanoma) cause elongation of the developing zebrafish embryo at 12 hpf, and severe developmental abnormalities at 24 and 48 hpf. In contrast, embryos expressing BRAFWT undergo normal development at all stages. No differences were detected in WT or disease allele expressing 4 hpf embryos. (B) Western blotting of zebrafish extracts reveals expression of the myc-tagged BRAF variants with the 9E10 antibody, and α-tubulin is a loading control.

Corina Anastasaki, et al. Hum Mol Genet. 2009 July 15;18(14):2543-2554.
5.
Figure 7.

Figure 7. From: Kinase-activating and kinase-impaired cardio-facio-cutaneous syndrome alleles have activity during zebrafish development and are sensitive to small molecule inhibitors.

Evaluation of CFC-disease variant in vivo activity and potential treatment. Schematic representation of the zebrafish-based approach designed to examine the in vivo significance of BRAF and MEK CFC disease mutations. (A) Microinjection of BRAF or MEK CFC variant mRNA into the single-cell zebrafish embryo promoted an elongated zebrafish embryo at 10 hpf that gives rise to an animal with severe development defects, including axis formation, and heart defects at 24 hpf (red arrow). (B) Treatment of CFC-microinjected embryos with inhibitors of the FGF-MAPK signalling pathway (green) restores normal development to the CFC-zebrafish embryo, possibly by restoring appropriate total levels of MAPK-signalling (green arrows).

Corina Anastasaki, et al. Hum Mol Genet. 2009 July 15;18(14):2543-2554.
6.
Figure 5.

Figure 5. From: Kinase-activating and kinase-impaired cardio-facio-cutaneous syndrome alleles have activity during zebrafish development and are sensitive to small molecule inhibitors.

Identification of a zebrafish-CFC treatment window. (A) Six CI-1040 treatments (A–F) and a control treatment (G) for zebrafish embryos expressing CFC variant BRAFQ257R reveal that all drug treatments restore normal development at 10.5 hpf (treatments A–F), whereas only treatments A and B restore normal development both at 10.5 and 27.5 hpf. Other drug treatments (C–F) cause additional developmental defects at 27.5 hpf, most notably, reduced posterior development. (B) Six additional drug treatments (A–F) and a control treatment (G) for zebrafish-CFC variant BRAFQ257R show that the 4.5–5.5 hpf developmental window is necessary for the prevention of zebrafish-CFC early phenotypes. The percentage of embryos displaying the phenotype is given at the lower right of each image (a minimal of n = 30/experiment). Blue bars represent embryo medium, whereas red bars represent CI-1040 treatment.

Corina Anastasaki, et al. Hum Mol Genet. 2009 July 15;18(14):2543-2554.
7.
Figure 2.

Figure 2. From: Kinase-activating and kinase-impaired cardio-facio-cutaneous syndrome alleles have activity during zebrafish development and are sensitive to small molecule inhibitors.

Analysis and treatment of kinase-activating and kinase-impaired BRAF and MEK disease variants. (A) RNA expression of BRAF CFC and melanoma variants, as well as engineered BRAF mutations, promotes an elongated embryo phenotype. RNA expression of wild-type BRAF has no effect on development. The developmental phenotypes caused by the BRAF variants are prevented by treatment with the MEK inhibitor, CI-1040 and the embryo has a normal rounded shape. (B) Western blotting of zebrafish lysates with anti-ERK and anti-phospho-ERK antibodies reveals a reduction in the ratio of phospho-ERK to total ERK protein in treated embryos, and α-tubulin is a loading control. (C) RNA expression of CFC MEK variants, the MEK1 constitutively activating (ΔN3DD) and the MEK2 kinase-inactivating K101M mutations promotes elongation in the developing embryo. Expression of wild-type MEK1 and MEK2 do not affect development. The developmental phenotypes caused by the MEK alleles are prevented by treatment with the MEK inhibitors, CI-1040 and PD0325901. (D and E) Western blotting of zebrafish lysates with anti-ERK and anti-phospho-ERK antibodies reveals downstream activity of phospho-ERK and confirms the potency of the MEK inhibitors, and α-tubulin is a loading control.

Corina Anastasaki, et al. Hum Mol Genet. 2009 July 15;18(14):2543-2554.

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