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  • 99
    Cell Signaling Technology Inc p 53
    P 53, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 27 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology p 53
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    Thermo Fisher plenti6 v5 p 53
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    Becton Dickinson p 53
    P 53, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    p 53  (KUBOTA)
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    KUBOTA p 53
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    Santa Cruz Biotechnology phosphor p 53
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    Novocastra monoclonal antibodies against p 53
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    Abcam rabbit polyclonal antibody p 53
    Rabbit Polyclonal Antibody P 53, supplied by Abcam, used in various techniques. Bioz Stars score: 90/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc human p 53 elisa kits
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    Santa Cruz Biotechnology p 53 primer antibodies
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    Addgene inc plenti6 v5 p 53 r249s
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    Thermo Fisher p53
    Upon genotoxic stress, p63 is stabilized in melanoma cells and partially relocates to the mitochondria. (A) Immunofluorescence microscopy of untreated melanocytes (Hema V3) and treatment with 50 mJ/cm 2 UVB (for 24 h) or 10 µM cisplatin (for 24 h). Induction of DNA damage and apoptosis assessed using γ-H2AX and cleaved caspase, respectively. DAPI was used to stain nuclei. Images are representative of three independent experiments. (B, top row) Untreated A375M cells contain low levels of endogenous p63 (detected by anti-p63 antibodies H129 and H137), which is largely nuclear. Significant up-regulation of p63 (green) in both nuclear and cytoplasmic compartments of A375M cells was observed upon treatment with 20 µM etoposide (second row), 2 µM paclitaxel (third row), and 2 µM doxorubicin (bottom row) for 6 h. DAPI was used to stain nuclei (blue). Images are representative of three independent experiments. (C) RT-PCR demonstrating up-regulation of TA and/or ΔN p63 expression in three melanoma cell lines, but not melanocytes upon treatment with 10 µM cisplatin for 24 h. GUS was used as a loading control. (D) Western blot of A375M cells treated with cisplatin and UVB. The molecular masses of exogenously transfected p63 plasmids in HEK 293T cells were used to interpret specific p63 isoforms affected by the treatment. Stabilization of both p63 isoforms and <t>p53</t> demonstrates a dose-dependent effect. (E) RT-PCR of TAp63 in A375M and WM1158 cell lines treated with 3 µM PLX4032 for 24 h. GUS was used as housekeeping gene for mRNA standardization. (F) Q-PCR of ΔNp63 expression in two BRAF mutated melanoma cell lines upon 24-h treatment with 3 µM PLX4032 or PLX4720. GUS was used as an endogenous control. Data show mean expression ± SD for at least three independent analyses performed in duplicates. (G) A375M cells labeled with MitoTracker Orange before treatment with 2 µM paclitaxel and immunofluorescence images shown at 6 and 9 h. DAPI was used to label nuclei. Images are representative of three independent experiments. (H) Western blot of WM1158 cells shows differential subcellular expression of p63 splice variants (TAp63α in the nuclear fraction and TAp63γ in the mitochondrial fraction). This was confirmed by comparing molecular masses with exogenously transfected p63 plasmids in HEK 293T cells (far left six lanes). Treatment with various chemotherapeutic agents, M2/N2 cisplatin, M3/N3 doxorubicin, M4/N4 paclitaxel, M5/N5 etoposide, and M6/N6 UVB, corresponding to mitochondrial (M) and nuclear (N) fractions of the same cells. p53 expression was analyzed using anti-p53 antibody (DO-1), and mtHsp70 and Lamin-A were used as markers for mitochondrial and nuclear protein loading, respectively. Western blot data are representative of cellular fractionation experiments performed in triplicate. (I) Transmission electron micrographs of immunogold labeling of p63 in mitochondria in A375M cells. Mitochondria are characterized by double membrane and cristae projections. (i) Negative control to confirm specificity comprises exclusion of primary antibody. (ii) Mitochondrion confirmed by electron-dense matrix surrounded by double membrane with cristae projections showing immunogold-labeled mtHsp70 localization within the mitochondrial matrix (black arrows). (iii–vi) Electron-dense mitochondria characterized by remarkably electron-dense mitochondrial matrix and cristae. Images demonstrate immunogold localization of p63 (using combination of anti-p63 antibodies H129 and H137) in cytosol, on mitochondrial membranes, and within mitochondrial matrix (white arrows). (J) Transmission electron micrographs of immunogold labeling of p63 in mitochondria (WM1158 cells). (i) Mitochondrion visualized by electron-dense matrix surrounded by double membrane with cristae projections showing immunogold-labeled mtHsp70 localization within the mitochondrial matrix (black arrows). (ii) Nuclear (labeled N) and extranuclear localization of p63. (iii) Electron-dense mitochondria characterized by electron-dense mitochondrial matrix and cristae. Image demonstrates immunogold localization of p63 (using combination of anti-p63 antibody H129) in transit to mitochondria and within mitochondrial matrix. (iv) Electron micrograph showing immunogold labeling of p63 (using H137 anti-p63 antibody) within electron-dense mitochondrion matrix (arrow), and cristae are visible. Images are representative of three independent experiments. Bars: (A, B, and G) 10 µm; (I [i–iii and v] and J [iii]) 100 nm; (I, iv and vi) 200 nm; (J, i, ii, and iv) 0.2 µm.
    P53, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 1906 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher p53 do1
    Upon genotoxic stress, p63 is stabilized in melanoma cells and partially relocates to the mitochondria. (A) Immunofluorescence microscopy of untreated melanocytes (Hema V3) and treatment with 50 mJ/cm 2 UVB (for 24 h) or 10 µM cisplatin (for 24 h). Induction of DNA damage and apoptosis assessed using γ-H2AX and cleaved caspase, respectively. DAPI was used to stain nuclei. Images are representative of three independent experiments. (B, top row) Untreated A375M cells contain low levels of endogenous p63 (detected by anti-p63 antibodies H129 and H137), which is largely nuclear. Significant up-regulation of p63 (green) in both nuclear and cytoplasmic compartments of A375M cells was observed upon treatment with 20 µM etoposide (second row), 2 µM paclitaxel (third row), and 2 µM doxorubicin (bottom row) for 6 h. DAPI was used to stain nuclei (blue). Images are representative of three independent experiments. (C) RT-PCR demonstrating up-regulation of TA and/or ΔN p63 expression in three melanoma cell lines, but not melanocytes upon treatment with 10 µM cisplatin for 24 h. GUS was used as a loading control. (D) Western blot of A375M cells treated with cisplatin and UVB. The molecular masses of exogenously transfected p63 plasmids in HEK 293T cells were used to interpret specific p63 isoforms affected by the treatment. Stabilization of both p63 isoforms and <t>p53</t> demonstrates a dose-dependent effect. (E) RT-PCR of TAp63 in A375M and WM1158 cell lines treated with 3 µM PLX4032 for 24 h. GUS was used as housekeeping gene for mRNA standardization. (F) Q-PCR of ΔNp63 expression in two BRAF mutated melanoma cell lines upon 24-h treatment with 3 µM PLX4032 or PLX4720. GUS was used as an endogenous control. Data show mean expression ± SD for at least three independent analyses performed in duplicates. (G) A375M cells labeled with MitoTracker Orange before treatment with 2 µM paclitaxel and immunofluorescence images shown at 6 and 9 h. DAPI was used to label nuclei. Images are representative of three independent experiments. (H) Western blot of WM1158 cells shows differential subcellular expression of p63 splice variants (TAp63α in the nuclear fraction and TAp63γ in the mitochondrial fraction). This was confirmed by comparing molecular masses with exogenously transfected p63 plasmids in HEK 293T cells (far left six lanes). Treatment with various chemotherapeutic agents, M2/N2 cisplatin, M3/N3 doxorubicin, M4/N4 paclitaxel, M5/N5 etoposide, and M6/N6 UVB, corresponding to mitochondrial (M) and nuclear (N) fractions of the same cells. p53 expression was analyzed using anti-p53 antibody (DO-1), and mtHsp70 and Lamin-A were used as markers for mitochondrial and nuclear protein loading, respectively. Western blot data are representative of cellular fractionation experiments performed in triplicate. (I) Transmission electron micrographs of immunogold labeling of p63 in mitochondria in A375M cells. Mitochondria are characterized by double membrane and cristae projections. (i) Negative control to confirm specificity comprises exclusion of primary antibody. (ii) Mitochondrion confirmed by electron-dense matrix surrounded by double membrane with cristae projections showing immunogold-labeled mtHsp70 localization within the mitochondrial matrix (black arrows). (iii–vi) Electron-dense mitochondria characterized by remarkably electron-dense mitochondrial matrix and cristae. Images demonstrate immunogold localization of p63 (using combination of anti-p63 antibodies H129 and H137) in cytosol, on mitochondrial membranes, and within mitochondrial matrix (white arrows). (J) Transmission electron micrographs of immunogold labeling of p63 in mitochondria (WM1158 cells). (i) Mitochondrion visualized by electron-dense matrix surrounded by double membrane with cristae projections showing immunogold-labeled mtHsp70 localization within the mitochondrial matrix (black arrows). (ii) Nuclear (labeled N) and extranuclear localization of p63. (iii) Electron-dense mitochondria characterized by electron-dense mitochondrial matrix and cristae. Image demonstrates immunogold localization of p63 (using combination of anti-p63 antibody H129) in transit to mitochondria and within mitochondrial matrix. (iv) Electron micrograph showing immunogold labeling of p63 (using H137 anti-p63 antibody) within electron-dense mitochondrion matrix (arrow), and cristae are visible. Images are representative of three independent experiments. Bars: (A, B, and G) 10 µm; (I [i–iii and v] and J [iii]) 100 nm; (I, iv and vi) 200 nm; (J, i, ii, and iv) 0.2 µm.
    P53 Do1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 88/100, based on 29 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    MOCAP p 53 co injected embryos
    Loss of CAP-G causes p53-mediated apoptosis within the retina . (A) Retinal expression of the proliferation marker pcna or of neurogenesis marker elval3 is not affected in cap-g s 105 mutants as detected by whole-mount in situ hybridizations at 3 dpf. (B) Transverse cryosections of embryonic retinae were stained against phosphorylated histone 3 which marks mitotic nuclei (red), TUNEL to detect apoptotic cells (green), and nuclei counterstained with DAPI (blue) at 24 hpf. Predominantly mitotic cells which divide at the ventricular side of the retina are apoptotic in cap-g s 105 mutants at 24 hpf. (C) Transverse vibratome sections of 4 dpf retinae stained against activated caspase 3 to detect apoptotic cells (red) and nuclei counterstained with propidium iodide (green). At this stage, proliferation is restricted to the CMZ. In cap-g s 105 mutants, cell death is restricted to the CMZ which indicates that proliferative cells are eliminated. (D) Transverse vibratome sections of embryonic retinae counterstained with phalloidin to visualize plexiform layers (red) and propidium iodide (green). cap-g s 105 mutants injected with MO <t>p</t> 53 show a rescue of retinal development and display correct retinal layering. (E) Quantification of cell numbers within different retinal cell layers. Propidium iodide stained transverse retinal sections were used to determine average counts for wild-type (n = 9 section planes, 5 embryos), cap-g s 105 mutants (n = 11 section planes, 7 embryos) or cap-g s 105 mutant/ p 53 morphants (n = 9 section planes, 6 embryos). The average sum of cap-g s 105 mutant retinal cells is reduced by 65% compared with wild-type. In comparison, the average sum of cap-g s 105 mutant/ p53 morphant retinal cells is reduced only by 43% compared with wild-type. Therefore, the severe reduction in retinal cell numbers is in part caused by p53-mediated apoptosis. Data represent average cell numbers per retina ± SD. T-test p-values for cell number differences in comparison to wild-type: ***, p
    P 53 Co Injected Embryos, supplied by MOCAP, used in various techniques. Bioz Stars score: 85/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Upon genotoxic stress, p63 is stabilized in melanoma cells and partially relocates to the mitochondria. (A) Immunofluorescence microscopy of untreated melanocytes (Hema V3) and treatment with 50 mJ/cm 2 UVB (for 24 h) or 10 µM cisplatin (for 24 h). Induction of DNA damage and apoptosis assessed using γ-H2AX and cleaved caspase, respectively. DAPI was used to stain nuclei. Images are representative of three independent experiments. (B, top row) Untreated A375M cells contain low levels of endogenous p63 (detected by anti-p63 antibodies H129 and H137), which is largely nuclear. Significant up-regulation of p63 (green) in both nuclear and cytoplasmic compartments of A375M cells was observed upon treatment with 20 µM etoposide (second row), 2 µM paclitaxel (third row), and 2 µM doxorubicin (bottom row) for 6 h. DAPI was used to stain nuclei (blue). Images are representative of three independent experiments. (C) RT-PCR demonstrating up-regulation of TA and/or ΔN p63 expression in three melanoma cell lines, but not melanocytes upon treatment with 10 µM cisplatin for 24 h. GUS was used as a loading control. (D) Western blot of A375M cells treated with cisplatin and UVB. The molecular masses of exogenously transfected p63 plasmids in HEK 293T cells were used to interpret specific p63 isoforms affected by the treatment. Stabilization of both p63 isoforms and p53 demonstrates a dose-dependent effect. (E) RT-PCR of TAp63 in A375M and WM1158 cell lines treated with 3 µM PLX4032 for 24 h. GUS was used as housekeeping gene for mRNA standardization. (F) Q-PCR of ΔNp63 expression in two BRAF mutated melanoma cell lines upon 24-h treatment with 3 µM PLX4032 or PLX4720. GUS was used as an endogenous control. Data show mean expression ± SD for at least three independent analyses performed in duplicates. (G) A375M cells labeled with MitoTracker Orange before treatment with 2 µM paclitaxel and immunofluorescence images shown at 6 and 9 h. DAPI was used to label nuclei. Images are representative of three independent experiments. (H) Western blot of WM1158 cells shows differential subcellular expression of p63 splice variants (TAp63α in the nuclear fraction and TAp63γ in the mitochondrial fraction). This was confirmed by comparing molecular masses with exogenously transfected p63 plasmids in HEK 293T cells (far left six lanes). Treatment with various chemotherapeutic agents, M2/N2 cisplatin, M3/N3 doxorubicin, M4/N4 paclitaxel, M5/N5 etoposide, and M6/N6 UVB, corresponding to mitochondrial (M) and nuclear (N) fractions of the same cells. p53 expression was analyzed using anti-p53 antibody (DO-1), and mtHsp70 and Lamin-A were used as markers for mitochondrial and nuclear protein loading, respectively. Western blot data are representative of cellular fractionation experiments performed in triplicate. (I) Transmission electron micrographs of immunogold labeling of p63 in mitochondria in A375M cells. Mitochondria are characterized by double membrane and cristae projections. (i) Negative control to confirm specificity comprises exclusion of primary antibody. (ii) Mitochondrion confirmed by electron-dense matrix surrounded by double membrane with cristae projections showing immunogold-labeled mtHsp70 localization within the mitochondrial matrix (black arrows). (iii–vi) Electron-dense mitochondria characterized by remarkably electron-dense mitochondrial matrix and cristae. Images demonstrate immunogold localization of p63 (using combination of anti-p63 antibodies H129 and H137) in cytosol, on mitochondrial membranes, and within mitochondrial matrix (white arrows). (J) Transmission electron micrographs of immunogold labeling of p63 in mitochondria (WM1158 cells). (i) Mitochondrion visualized by electron-dense matrix surrounded by double membrane with cristae projections showing immunogold-labeled mtHsp70 localization within the mitochondrial matrix (black arrows). (ii) Nuclear (labeled N) and extranuclear localization of p63. (iii) Electron-dense mitochondria characterized by electron-dense mitochondrial matrix and cristae. Image demonstrates immunogold localization of p63 (using combination of anti-p63 antibody H129) in transit to mitochondria and within mitochondrial matrix. (iv) Electron micrograph showing immunogold labeling of p63 (using H137 anti-p63 antibody) within electron-dense mitochondrion matrix (arrow), and cristae are visible. Images are representative of three independent experiments. Bars: (A, B, and G) 10 µm; (I [i–iii and v] and J [iii]) 100 nm; (I, iv and vi) 200 nm; (J, i, ii, and iv) 0.2 µm.

    Journal: The Journal of Experimental Medicine

    Article Title: p63 is an alternative p53 repressor in melanoma that confers chemoresistance and a poor prognosis

    doi: 10.1084/jem.20121439

    Figure Lengend Snippet: Upon genotoxic stress, p63 is stabilized in melanoma cells and partially relocates to the mitochondria. (A) Immunofluorescence microscopy of untreated melanocytes (Hema V3) and treatment with 50 mJ/cm 2 UVB (for 24 h) or 10 µM cisplatin (for 24 h). Induction of DNA damage and apoptosis assessed using γ-H2AX and cleaved caspase, respectively. DAPI was used to stain nuclei. Images are representative of three independent experiments. (B, top row) Untreated A375M cells contain low levels of endogenous p63 (detected by anti-p63 antibodies H129 and H137), which is largely nuclear. Significant up-regulation of p63 (green) in both nuclear and cytoplasmic compartments of A375M cells was observed upon treatment with 20 µM etoposide (second row), 2 µM paclitaxel (third row), and 2 µM doxorubicin (bottom row) for 6 h. DAPI was used to stain nuclei (blue). Images are representative of three independent experiments. (C) RT-PCR demonstrating up-regulation of TA and/or ΔN p63 expression in three melanoma cell lines, but not melanocytes upon treatment with 10 µM cisplatin for 24 h. GUS was used as a loading control. (D) Western blot of A375M cells treated with cisplatin and UVB. The molecular masses of exogenously transfected p63 plasmids in HEK 293T cells were used to interpret specific p63 isoforms affected by the treatment. Stabilization of both p63 isoforms and p53 demonstrates a dose-dependent effect. (E) RT-PCR of TAp63 in A375M and WM1158 cell lines treated with 3 µM PLX4032 for 24 h. GUS was used as housekeeping gene for mRNA standardization. (F) Q-PCR of ΔNp63 expression in two BRAF mutated melanoma cell lines upon 24-h treatment with 3 µM PLX4032 or PLX4720. GUS was used as an endogenous control. Data show mean expression ± SD for at least three independent analyses performed in duplicates. (G) A375M cells labeled with MitoTracker Orange before treatment with 2 µM paclitaxel and immunofluorescence images shown at 6 and 9 h. DAPI was used to label nuclei. Images are representative of three independent experiments. (H) Western blot of WM1158 cells shows differential subcellular expression of p63 splice variants (TAp63α in the nuclear fraction and TAp63γ in the mitochondrial fraction). This was confirmed by comparing molecular masses with exogenously transfected p63 plasmids in HEK 293T cells (far left six lanes). Treatment with various chemotherapeutic agents, M2/N2 cisplatin, M3/N3 doxorubicin, M4/N4 paclitaxel, M5/N5 etoposide, and M6/N6 UVB, corresponding to mitochondrial (M) and nuclear (N) fractions of the same cells. p53 expression was analyzed using anti-p53 antibody (DO-1), and mtHsp70 and Lamin-A were used as markers for mitochondrial and nuclear protein loading, respectively. Western blot data are representative of cellular fractionation experiments performed in triplicate. (I) Transmission electron micrographs of immunogold labeling of p63 in mitochondria in A375M cells. Mitochondria are characterized by double membrane and cristae projections. (i) Negative control to confirm specificity comprises exclusion of primary antibody. (ii) Mitochondrion confirmed by electron-dense matrix surrounded by double membrane with cristae projections showing immunogold-labeled mtHsp70 localization within the mitochondrial matrix (black arrows). (iii–vi) Electron-dense mitochondria characterized by remarkably electron-dense mitochondrial matrix and cristae. Images demonstrate immunogold localization of p63 (using combination of anti-p63 antibodies H129 and H137) in cytosol, on mitochondrial membranes, and within mitochondrial matrix (white arrows). (J) Transmission electron micrographs of immunogold labeling of p63 in mitochondria (WM1158 cells). (i) Mitochondrion visualized by electron-dense matrix surrounded by double membrane with cristae projections showing immunogold-labeled mtHsp70 localization within the mitochondrial matrix (black arrows). (ii) Nuclear (labeled N) and extranuclear localization of p63. (iii) Electron-dense mitochondria characterized by electron-dense mitochondrial matrix and cristae. Image demonstrates immunogold localization of p63 (using combination of anti-p63 antibody H129) in transit to mitochondria and within mitochondrial matrix. (iv) Electron micrograph showing immunogold labeling of p63 (using H137 anti-p63 antibody) within electron-dense mitochondrion matrix (arrow), and cristae are visible. Images are representative of three independent experiments. Bars: (A, B, and G) 10 µm; (I [i–iii and v] and J [iii]) 100 nm; (I, iv and vi) 200 nm; (J, i, ii, and iv) 0.2 µm.

    Article Snippet: For p53 knockdown, A375M cells were transfected with the stealth RNAi siRNA for p53 (ID 000546; Invitrogen; ).

    Techniques: Immunofluorescence, Microscopy, Staining, Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot, Transfection, Polymerase Chain Reaction, Labeling, Cell Fractionation, Transmission Assay, Negative Control

    Endogenous p63 prevents p53 nuclear stabilization and requires p53 to translocate into the mitochondria after DNA damage. (A, left) Protein expression of p53, MDM2, and MDM4 in A375M cells depleted of p53 (transient transfection 48 h). GAPDH was used as a loading marker. (right) Profile of relative changes in total p63 expression using flow cytometry fractionation technique in a p53-null melanoma cell line (A375M si-p53) compared with the parental control cell line (A375M si-Ctrl) upon treatment with paclitaxel and etoposide (6 h). (B and C) Comparison of nuclear (B) and mitochondrial (C) p63 protein stabilization in A375M si-p53 versus the parental cell line expressing WT-p53 (A375M si-Ctrl). Data show mean protein expression ± SEM for at least three independent experiments performed in duplicate. (D) Histograms showing relative changes in p53 expression in A375M scramble cells and A375M sh-p63 cells upon treatment with paclitaxel or etoposide (6 h); total (left), nuclear (middle), and mitochondrial (right) p53 in p63-depleted cells compared with scramble-control. (A and D) Data show mean protein expression ± SEM for at least three independent experiments performed in duplicate. (E) Western blot of total and phosphorylated p53, MDM2, and MDM4 derived from nuclear extracts of scramble-A375M cells and sh-p63 cells after treatment with paclitaxel (6 h). PCNA nuclear marker was used as a loading marker. (F) Immunoprecipitation (IP) of endogenous p53 in untreated A375M cells reveals physiological interaction with ΔNp63β. Input: A375M total lysates. HEK293 cells were transfected with ΔNp63 β as a loading marker control. WB, Western blot.

    Journal: The Journal of Experimental Medicine

    Article Title: p63 is an alternative p53 repressor in melanoma that confers chemoresistance and a poor prognosis

    doi: 10.1084/jem.20121439

    Figure Lengend Snippet: Endogenous p63 prevents p53 nuclear stabilization and requires p53 to translocate into the mitochondria after DNA damage. (A, left) Protein expression of p53, MDM2, and MDM4 in A375M cells depleted of p53 (transient transfection 48 h). GAPDH was used as a loading marker. (right) Profile of relative changes in total p63 expression using flow cytometry fractionation technique in a p53-null melanoma cell line (A375M si-p53) compared with the parental control cell line (A375M si-Ctrl) upon treatment with paclitaxel and etoposide (6 h). (B and C) Comparison of nuclear (B) and mitochondrial (C) p63 protein stabilization in A375M si-p53 versus the parental cell line expressing WT-p53 (A375M si-Ctrl). Data show mean protein expression ± SEM for at least three independent experiments performed in duplicate. (D) Histograms showing relative changes in p53 expression in A375M scramble cells and A375M sh-p63 cells upon treatment with paclitaxel or etoposide (6 h); total (left), nuclear (middle), and mitochondrial (right) p53 in p63-depleted cells compared with scramble-control. (A and D) Data show mean protein expression ± SEM for at least three independent experiments performed in duplicate. (E) Western blot of total and phosphorylated p53, MDM2, and MDM4 derived from nuclear extracts of scramble-A375M cells and sh-p63 cells after treatment with paclitaxel (6 h). PCNA nuclear marker was used as a loading marker. (F) Immunoprecipitation (IP) of endogenous p53 in untreated A375M cells reveals physiological interaction with ΔNp63β. Input: A375M total lysates. HEK293 cells were transfected with ΔNp63 β as a loading marker control. WB, Western blot.

    Article Snippet: For p53 knockdown, A375M cells were transfected with the stealth RNAi siRNA for p53 (ID 000546; Invitrogen; ).

    Techniques: Expressing, Transfection, Marker, Flow Cytometry, Cytometry, Fractionation, Western Blot, Derivative Assay, Immunoprecipitation

    Depletion of p63 by shRNA-p63 clones increases chemosensitivity. (A) Pictorial representation of TAp63 (top) and ΔNp63 (bottom) genes demonstrating targeted sequences used by various RNAi oligonucleotides (1, 2, and 3) designed to target regions in both TA and ΔN isoforms of p63. (B) Q-PCR (left) and Western blot (right) demonstrating knockdown of TAp63 gene and protein achieved in three shRNA-p63 clones. GUS was used as endogenous comparator for Q-PCR and GAPDH for Western blot. Data show mean ± SD of three independent experiments. (C) Percentage of apoptotic cells (Annexin V positive) displayed as mean ± SEM for three independent experiments performed in duplicate. Apoptosis in WM1158 shRNA-p63 cells compared with shRNA-scramble cells upon treatment with 10 µM cisplatin, 2 µM paclitaxel or 10 µM etoposide, and 200 µM dacarbazine. (D) Western blot of cytosolic fractions of WM1158 upon treatment with paclitaxel or etoposide (16 h) in cells with scramble-shRNA and those depleted of p63 using sh-RNA. (E, left) Q-PCR of cells transfected with three different shRNA-p63 sequences demonstrates significant reduction of ΔNp63 in A375M cells for all three when compared with shRNA-scramble–transfected cells. Data show mean expression ± SD for at least three independent analyses performed in duplicates. (right) Western blot of A375M cells demonstrates significant silencing of ΔNp63 protein levels (the predominantly expressed isoform) by shRNA p63 clones 1 and 2. (F) Percentage of apoptotic cells (Annexin V positive) displayed as mean ± SEM for three independent experiments performed in duplicate. Apoptosis in A375M shRNA-p63 cells compared with shRNA-scramble cells upon treatment with 10 µM cisplatin, 2 µM paclitaxel or 10 µM etoposide, and 200 µM dacarbazine. (G) Protein expression of p53 and selected downstream targets in cells depleted of p63 exposed to cisplatin, etoposide, or paclitaxel treatment. (H) Protein expression of p53 and selected downstream targets in A375M cells depleted of p63 upon treatment with paclitaxel. (I and J) Apoptosis in WM1158 shRNA-p63 (I) and A375M shRNA-p63 (J) cells compared with shRNA-scramble cells upon 24-h treatment with two BRAF inhibitors (3 µM PLX4032 or PLX4720). Percentage of apoptotic cells (Annexin V positive) is displayed as mean ± SEM for three independent experiments performed in duplicate. (K) Protein expression of selected BRAF downstream targets and p53 regulators in A375M cells depleted of p63 upon treatment with 3 µM PLX4032 or PLX4720 for 24 h.

    Journal: The Journal of Experimental Medicine

    Article Title: p63 is an alternative p53 repressor in melanoma that confers chemoresistance and a poor prognosis

    doi: 10.1084/jem.20121439

    Figure Lengend Snippet: Depletion of p63 by shRNA-p63 clones increases chemosensitivity. (A) Pictorial representation of TAp63 (top) and ΔNp63 (bottom) genes demonstrating targeted sequences used by various RNAi oligonucleotides (1, 2, and 3) designed to target regions in both TA and ΔN isoforms of p63. (B) Q-PCR (left) and Western blot (right) demonstrating knockdown of TAp63 gene and protein achieved in three shRNA-p63 clones. GUS was used as endogenous comparator for Q-PCR and GAPDH for Western blot. Data show mean ± SD of three independent experiments. (C) Percentage of apoptotic cells (Annexin V positive) displayed as mean ± SEM for three independent experiments performed in duplicate. Apoptosis in WM1158 shRNA-p63 cells compared with shRNA-scramble cells upon treatment with 10 µM cisplatin, 2 µM paclitaxel or 10 µM etoposide, and 200 µM dacarbazine. (D) Western blot of cytosolic fractions of WM1158 upon treatment with paclitaxel or etoposide (16 h) in cells with scramble-shRNA and those depleted of p63 using sh-RNA. (E, left) Q-PCR of cells transfected with three different shRNA-p63 sequences demonstrates significant reduction of ΔNp63 in A375M cells for all three when compared with shRNA-scramble–transfected cells. Data show mean expression ± SD for at least three independent analyses performed in duplicates. (right) Western blot of A375M cells demonstrates significant silencing of ΔNp63 protein levels (the predominantly expressed isoform) by shRNA p63 clones 1 and 2. (F) Percentage of apoptotic cells (Annexin V positive) displayed as mean ± SEM for three independent experiments performed in duplicate. Apoptosis in A375M shRNA-p63 cells compared with shRNA-scramble cells upon treatment with 10 µM cisplatin, 2 µM paclitaxel or 10 µM etoposide, and 200 µM dacarbazine. (G) Protein expression of p53 and selected downstream targets in cells depleted of p63 exposed to cisplatin, etoposide, or paclitaxel treatment. (H) Protein expression of p53 and selected downstream targets in A375M cells depleted of p63 upon treatment with paclitaxel. (I and J) Apoptosis in WM1158 shRNA-p63 (I) and A375M shRNA-p63 (J) cells compared with shRNA-scramble cells upon 24-h treatment with two BRAF inhibitors (3 µM PLX4032 or PLX4720). Percentage of apoptotic cells (Annexin V positive) is displayed as mean ± SEM for three independent experiments performed in duplicate. (K) Protein expression of selected BRAF downstream targets and p53 regulators in A375M cells depleted of p63 upon treatment with 3 µM PLX4032 or PLX4720 for 24 h.

    Article Snippet: For p53 knockdown, A375M cells were transfected with the stealth RNAi siRNA for p53 (ID 000546; Invitrogen; ).

    Techniques: shRNA, Clone Assay, Polymerase Chain Reaction, Western Blot, Transfection, Expressing

    p63 is expressed in melanoma cell lines and melanoma tissue samples. (A) TP63 expression in human normal skin cellular components (RT-PCR). Controls included omission of cDNA. GAPDH was used as a loading control. (B) Q-PCR of TAp63 (39%, 13/33; top) and ΔNp63 (51%, 17/33; bottom) in panel of established primary melanoma and metastatic melanoma cell lines. The bars show the folds of p63 mean expression ± SD compared with mean expression of TP63 / GUS in five primary melanocyte cultures (NHEM1, NHEM2, HEMa 3, HEMa V3, and HEMa V4). Measurements have been performed at least three times for each cell line at different passages. Dotted lines mark threefold increase in gene expression compared with mean expression in melanocyte cultures. GUS was used as an endogenous control. (C) 80 µg protein lysates from primary melanocyte cultures (NHEM and Hema) and melanoma cell lines probed for p63 (using anti-p63 antibody AB-4 which detects all isoforms of p63). GAPDH was used as loading control. (D) Analysis of mean expression ± SD of TP53 family genes in melanoma tissue samples using gene microarray (Agilent Technologies; Scatolini et al., 2010 ). VGP, vertical growth phase; Metastatic, melanoma metastases. (E) Immunofluorescence microscopy demonstrating expression of p63 in primary cutaneous melanomas (mTMA). (left) Primary melanoma from left arm (81-yr-old female). (right) Primary melanoma with heavy melanin deposition from right thumb (62-yr-old female). Panels described left to right, top to bottom: hematoxylin and eosin (H E) labeling of melanoma, DAPI staining nuclei of melanoma cells, HMB-45 labeling melanoma cells, anti-p63 antibodies (H129/H137) labeling cells within tumor for p63, and merged image demonstrating coexpression of HMB-45 and p63 (yellow). Higher magnification confirms melanoma cells express p63 (yellow). Bars: (H E thru Merge) 100 µm; (Merge, zoom) 50 µm.

    Journal: The Journal of Experimental Medicine

    Article Title: p63 is an alternative p53 repressor in melanoma that confers chemoresistance and a poor prognosis

    doi: 10.1084/jem.20121439

    Figure Lengend Snippet: p63 is expressed in melanoma cell lines and melanoma tissue samples. (A) TP63 expression in human normal skin cellular components (RT-PCR). Controls included omission of cDNA. GAPDH was used as a loading control. (B) Q-PCR of TAp63 (39%, 13/33; top) and ΔNp63 (51%, 17/33; bottom) in panel of established primary melanoma and metastatic melanoma cell lines. The bars show the folds of p63 mean expression ± SD compared with mean expression of TP63 / GUS in five primary melanocyte cultures (NHEM1, NHEM2, HEMa 3, HEMa V3, and HEMa V4). Measurements have been performed at least three times for each cell line at different passages. Dotted lines mark threefold increase in gene expression compared with mean expression in melanocyte cultures. GUS was used as an endogenous control. (C) 80 µg protein lysates from primary melanocyte cultures (NHEM and Hema) and melanoma cell lines probed for p63 (using anti-p63 antibody AB-4 which detects all isoforms of p63). GAPDH was used as loading control. (D) Analysis of mean expression ± SD of TP53 family genes in melanoma tissue samples using gene microarray (Agilent Technologies; Scatolini et al., 2010 ). VGP, vertical growth phase; Metastatic, melanoma metastases. (E) Immunofluorescence microscopy demonstrating expression of p63 in primary cutaneous melanomas (mTMA). (left) Primary melanoma from left arm (81-yr-old female). (right) Primary melanoma with heavy melanin deposition from right thumb (62-yr-old female). Panels described left to right, top to bottom: hematoxylin and eosin (H E) labeling of melanoma, DAPI staining nuclei of melanoma cells, HMB-45 labeling melanoma cells, anti-p63 antibodies (H129/H137) labeling cells within tumor for p63, and merged image demonstrating coexpression of HMB-45 and p63 (yellow). Higher magnification confirms melanoma cells express p63 (yellow). Bars: (H E thru Merge) 100 µm; (Merge, zoom) 50 µm.

    Article Snippet: For p53 knockdown, A375M cells were transfected with the stealth RNAi siRNA for p53 (ID 000546; Invitrogen; ).

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Microarray, Immunofluorescence, Microscopy, Labeling, Staining

    Kaplan-Meier analysis of ( A ) OS and ( B ) PFS in all participants by p53 Arg72Pro genotype.

    Journal:

    Article Title: p53 Arg72Pro and MDM2 T309G Polymorphisms, Histology, and Esophageal Cancer Prognosis

    doi: 10.1158/1078-0432.CCR-08-3120

    Figure Lengend Snippet: Kaplan-Meier analysis of ( A ) OS and ( B ) PFS in all participants by p53 Arg72Pro genotype.

    Article Snippet: The p53 Arg72Pro (rs1042522) and MDM2 T309G (rs2279744) SNPs were genotyped using the TaqMan assay and a 384-well ABI 7900HT Sequence Detection System (Applied Biosystems).

    Techniques:

    Characterization of protein adhesion on WE by using fluorescence microscopy. Representative pictures of electrodes coated with fluorescently labeled p53 protein (right) compared to bare materials (left), both acquired with a 10X magnification on C (top) and Pt (bottom).

    Journal: Scientific Reports

    Article Title: Electrochemical detection of different p53 conformations by using nanostructured surfaces

    doi: 10.1038/s41598-019-53994-6

    Figure Lengend Snippet: Characterization of protein adhesion on WE by using fluorescence microscopy. Representative pictures of electrodes coated with fluorescently labeled p53 protein (right) compared to bare materials (left), both acquired with a 10X magnification on C (top) and Pt (bottom).

    Article Snippet: A solution containing 6 µg/ml p53 protein was incubated in the dark for 15 min with a fluorescent labeling reagent (Qubit protein assay organic dye, Invitrogen, Buenos Aires, Argentina), and then it was coated on the electrode surface and incubated overnight at 4 °C.

    Techniques: Fluorescence, Microscopy, Labeling

    Label-based calibration of denatured p53 performed using the ASV protocol with PAb240 on SPEs of different materials. The plots show the calibration comparing bare and nanostructured C- and Pt-based materials. For Pt, physical and chemical adsorption (Pt Act) are compared.

    Journal: Scientific Reports

    Article Title: Electrochemical detection of different p53 conformations by using nanostructured surfaces

    doi: 10.1038/s41598-019-53994-6

    Figure Lengend Snippet: Label-based calibration of denatured p53 performed using the ASV protocol with PAb240 on SPEs of different materials. The plots show the calibration comparing bare and nanostructured C- and Pt-based materials. For Pt, physical and chemical adsorption (Pt Act) are compared.

    Article Snippet: A solution containing 6 µg/ml p53 protein was incubated in the dark for 15 min with a fluorescent labeling reagent (Qubit protein assay organic dye, Invitrogen, Buenos Aires, Argentina), and then it was coated on the electrode surface and incubated overnight at 4 °C.

    Techniques: Adsorption, Activated Clotting Time Assay

    Label-free calibration of denatured p53 using CV on SPEs of different materials. The calibration plots compare bare and nanostructured C- and Pt-based materials. For Pt, physical and chemical adsorption (Pt Act) are compared.

    Journal: Scientific Reports

    Article Title: Electrochemical detection of different p53 conformations by using nanostructured surfaces

    doi: 10.1038/s41598-019-53994-6

    Figure Lengend Snippet: Label-free calibration of denatured p53 using CV on SPEs of different materials. The calibration plots compare bare and nanostructured C- and Pt-based materials. For Pt, physical and chemical adsorption (Pt Act) are compared.

    Article Snippet: A solution containing 6 µg/ml p53 protein was incubated in the dark for 15 min with a fluorescent labeling reagent (Qubit protein assay organic dye, Invitrogen, Buenos Aires, Argentina), and then it was coated on the electrode surface and incubated overnight at 4 °C.

    Techniques: Adsorption, Activated Clotting Time Assay

    Label-based qualitative detection of different p53 redox products using silver stripping voltammetry. Peaks resulting from protein quantification by the well-known conformationally altered antibody Ab240 are represented.

    Journal: Scientific Reports

    Article Title: Electrochemical detection of different p53 conformations by using nanostructured surfaces

    doi: 10.1038/s41598-019-53994-6

    Figure Lengend Snippet: Label-based qualitative detection of different p53 redox products using silver stripping voltammetry. Peaks resulting from protein quantification by the well-known conformationally altered antibody Ab240 are represented.

    Article Snippet: A solution containing 6 µg/ml p53 protein was incubated in the dark for 15 min with a fluorescent labeling reagent (Qubit protein assay organic dye, Invitrogen, Buenos Aires, Argentina), and then it was coated on the electrode surface and incubated overnight at 4 °C.

    Techniques: Stripping Membranes

    ( A–F ) CVs of control SPEs and SPEs coated with denatured p53, with and without nanostructures. ( A ) C vs ( B ) MWCNTs, ( C ) Pt vs ( D ) Pt NPTs act, ( E ) Pt vs ( F ) Pt NPTs), ( G ). Comparison between peaks highlighted in fig. ( A – C ), enhanced subtracting the baseline from the signal, to compare the effective contribute given by the same concentration of denatured p53 coated on different materials.

    Journal: Scientific Reports

    Article Title: Electrochemical detection of different p53 conformations by using nanostructured surfaces

    doi: 10.1038/s41598-019-53994-6

    Figure Lengend Snippet: ( A–F ) CVs of control SPEs and SPEs coated with denatured p53, with and without nanostructures. ( A ) C vs ( B ) MWCNTs, ( C ) Pt vs ( D ) Pt NPTs act, ( E ) Pt vs ( F ) Pt NPTs), ( G ). Comparison between peaks highlighted in fig. ( A – C ), enhanced subtracting the baseline from the signal, to compare the effective contribute given by the same concentration of denatured p53 coated on different materials.

    Article Snippet: A solution containing 6 µg/ml p53 protein was incubated in the dark for 15 min with a fluorescent labeling reagent (Qubit protein assay organic dye, Invitrogen, Buenos Aires, Argentina), and then it was coated on the electrode surface and incubated overnight at 4 °C.

    Techniques: Activated Clotting Time Assay, Concentration Assay

    Electrochemical detection of different p53 redox products. On the left, label-free approach, a comparison between CVs performed coating SPEs with wild-type (black) , nitrated (orange) ; oxidized (green) and denatured (blu e ) p53 . The amino acid residues available for oxidative/nitrosative modifications are represented as R.

    Journal: Scientific Reports

    Article Title: Electrochemical detection of different p53 conformations by using nanostructured surfaces

    doi: 10.1038/s41598-019-53994-6

    Figure Lengend Snippet: Electrochemical detection of different p53 redox products. On the left, label-free approach, a comparison between CVs performed coating SPEs with wild-type (black) , nitrated (orange) ; oxidized (green) and denatured (blu e ) p53 . The amino acid residues available for oxidative/nitrosative modifications are represented as R.

    Article Snippet: A solution containing 6 µg/ml p53 protein was incubated in the dark for 15 min with a fluorescent labeling reagent (Qubit protein assay organic dye, Invitrogen, Buenos Aires, Argentina), and then it was coated on the electrode surface and incubated overnight at 4 °C.

    Techniques:

    Loss of CAP-G causes p53-mediated apoptosis within the retina . (A) Retinal expression of the proliferation marker pcna or of neurogenesis marker elval3 is not affected in cap-g s 105 mutants as detected by whole-mount in situ hybridizations at 3 dpf. (B) Transverse cryosections of embryonic retinae were stained against phosphorylated histone 3 which marks mitotic nuclei (red), TUNEL to detect apoptotic cells (green), and nuclei counterstained with DAPI (blue) at 24 hpf. Predominantly mitotic cells which divide at the ventricular side of the retina are apoptotic in cap-g s 105 mutants at 24 hpf. (C) Transverse vibratome sections of 4 dpf retinae stained against activated caspase 3 to detect apoptotic cells (red) and nuclei counterstained with propidium iodide (green). At this stage, proliferation is restricted to the CMZ. In cap-g s 105 mutants, cell death is restricted to the CMZ which indicates that proliferative cells are eliminated. (D) Transverse vibratome sections of embryonic retinae counterstained with phalloidin to visualize plexiform layers (red) and propidium iodide (green). cap-g s 105 mutants injected with MO p 53 show a rescue of retinal development and display correct retinal layering. (E) Quantification of cell numbers within different retinal cell layers. Propidium iodide stained transverse retinal sections were used to determine average counts for wild-type (n = 9 section planes, 5 embryos), cap-g s 105 mutants (n = 11 section planes, 7 embryos) or cap-g s 105 mutant/ p 53 morphants (n = 9 section planes, 6 embryos). The average sum of cap-g s 105 mutant retinal cells is reduced by 65% compared with wild-type. In comparison, the average sum of cap-g s 105 mutant/ p53 morphant retinal cells is reduced only by 43% compared with wild-type. Therefore, the severe reduction in retinal cell numbers is in part caused by p53-mediated apoptosis. Data represent average cell numbers per retina ± SD. T-test p-values for cell number differences in comparison to wild-type: ***, p

    Journal: BMC Developmental Biology

    Article Title: Non-SMC condensin I complex proteins control chromosome segregation and survival of proliferating cells in the zebrafish neural retina

    doi: 10.1186/1471-213X-9-40

    Figure Lengend Snippet: Loss of CAP-G causes p53-mediated apoptosis within the retina . (A) Retinal expression of the proliferation marker pcna or of neurogenesis marker elval3 is not affected in cap-g s 105 mutants as detected by whole-mount in situ hybridizations at 3 dpf. (B) Transverse cryosections of embryonic retinae were stained against phosphorylated histone 3 which marks mitotic nuclei (red), TUNEL to detect apoptotic cells (green), and nuclei counterstained with DAPI (blue) at 24 hpf. Predominantly mitotic cells which divide at the ventricular side of the retina are apoptotic in cap-g s 105 mutants at 24 hpf. (C) Transverse vibratome sections of 4 dpf retinae stained against activated caspase 3 to detect apoptotic cells (red) and nuclei counterstained with propidium iodide (green). At this stage, proliferation is restricted to the CMZ. In cap-g s 105 mutants, cell death is restricted to the CMZ which indicates that proliferative cells are eliminated. (D) Transverse vibratome sections of embryonic retinae counterstained with phalloidin to visualize plexiform layers (red) and propidium iodide (green). cap-g s 105 mutants injected with MO p 53 show a rescue of retinal development and display correct retinal layering. (E) Quantification of cell numbers within different retinal cell layers. Propidium iodide stained transverse retinal sections were used to determine average counts for wild-type (n = 9 section planes, 5 embryos), cap-g s 105 mutants (n = 11 section planes, 7 embryos) or cap-g s 105 mutant/ p 53 morphants (n = 9 section planes, 6 embryos). The average sum of cap-g s 105 mutant retinal cells is reduced by 65% compared with wild-type. In comparison, the average sum of cap-g s 105 mutant/ p53 morphant retinal cells is reduced only by 43% compared with wild-type. Therefore, the severe reduction in retinal cell numbers is in part caused by p53-mediated apoptosis. Data represent average cell numbers per retina ± SD. T-test p-values for cell number differences in comparison to wild-type: ***, p

    Article Snippet: The same phenotype was observed for MOcap-g +p 53 co-injected embryos (not shown).

    Techniques: Expressing, Marker, In Situ, Staining, TUNEL Assay, Injection, Mutagenesis

    Defective sister chromatid separation and aberrant nuclear sizes and shapes upon loss of Cap-G . (A) Selected images from a timelapse recording of mitotic divisions at gastrula stages analyzed in Tg [H2A::GFP] transgenic embryos. cap-g morphant embryos display defective sister chromatid separation and abnormal chromatid morphology during anaphase. (B) Selected images from a timelapse analysis of mitotic divisions within the ventricular zone of the neural tube at 32 hpf. Progression from prometaphase, when condensed chromosomes are visible, to anaphase is significantly delayed in cap-g s 105 mutants. (C) Transverse vibratome sections through retinae counter-stained with propidium iodide reveal aberrant nuclear sizes and morphologies within cap-g s 105 mutants at 3 dpf. The strict retinal layering into inner INL and PRL is not recognizable. Red asterisk indicates decondensed nucleus with chromatid bridge/non-disjunction event. Similarly, the non-neural tail region of cap-g s 105 mutants contains cells with incomplete separation of chromatids (red asterisk). (D) In the wild-type retina, most nuclei in the photoreceptor layer have an elongated appearance. In cap-g s 105 mutant or cap-g s 105 mutant/ p 53 morphant retinae, most PRL nuclei fail to elongate. Data represent mean ± SD, n ≥ 115 for each retinal layer and genotype; **, p

    Journal: BMC Developmental Biology

    Article Title: Non-SMC condensin I complex proteins control chromosome segregation and survival of proliferating cells in the zebrafish neural retina

    doi: 10.1186/1471-213X-9-40

    Figure Lengend Snippet: Defective sister chromatid separation and aberrant nuclear sizes and shapes upon loss of Cap-G . (A) Selected images from a timelapse recording of mitotic divisions at gastrula stages analyzed in Tg [H2A::GFP] transgenic embryos. cap-g morphant embryos display defective sister chromatid separation and abnormal chromatid morphology during anaphase. (B) Selected images from a timelapse analysis of mitotic divisions within the ventricular zone of the neural tube at 32 hpf. Progression from prometaphase, when condensed chromosomes are visible, to anaphase is significantly delayed in cap-g s 105 mutants. (C) Transverse vibratome sections through retinae counter-stained with propidium iodide reveal aberrant nuclear sizes and morphologies within cap-g s 105 mutants at 3 dpf. The strict retinal layering into inner INL and PRL is not recognizable. Red asterisk indicates decondensed nucleus with chromatid bridge/non-disjunction event. Similarly, the non-neural tail region of cap-g s 105 mutants contains cells with incomplete separation of chromatids (red asterisk). (D) In the wild-type retina, most nuclei in the photoreceptor layer have an elongated appearance. In cap-g s 105 mutant or cap-g s 105 mutant/ p 53 morphant retinae, most PRL nuclei fail to elongate. Data represent mean ± SD, n ≥ 115 for each retinal layer and genotype; **, p

    Article Snippet: The same phenotype was observed for MOcap-g +p 53 co-injected embryos (not shown).

    Techniques: Transgenic Assay, Staining, Mutagenesis