plasmids expressing p53 isoforms  (Promega)

Journal: Cancers

Article Title: Altered Expression of Shorter p53 Family Isoforms Can Impact Melanoma Aggressiveness

doi: 10.3390/cancers13205231

Figure Lengend Snippet: The gene expression of p53/p73 isoforms in melanoma cell lines. ( A ) Expression of the TP53 gene in the melanoma cell lines. The bars show relative expression levels of TP53 isoforms analyzed by pre-amplification followed by qPCR, normalized to the expression level of total TP53 . The cell lines are grouped according to their p53/BRAF mutational status. ( B ) Heatmap display of relative TP53 isoforms’ expression relative to the values of all isoforms (corresponding to the panel 1A) in each cell line; green color indicates the lowest, while red color the highest gene expression. Grey color indicates lack of expression. Isoform names written in black indicate BRAF wt, and in red, BRAF mt. ( C ) Expression of the TP73 gene in the melanoma cell lines. Relative expression levels of TP73 isoforms were analyzed by qPCR. The expression was normalized to the geometric mean of GUSB and TBP. Results are presented on a negative log scale. ( D ) Heatmap display of relative ΔNp73 and TAp73 expression (corresponding to the panel 1C left) in each cell line, and display of relative ΔEx2/3p73 expression (corresponding to the panel 1C right); green color indicates the lowest, while red color the highest gene expression. Grey color indicates lack of expression. Isoform names written in black indicate BRAF wt, and in red, BRAF mt. Presented are the averages of two independently performed experiments ± SD. Two-way ANOVA with Tukey’s multiple comparison test was used to determine differences in gene expression between groups based on mutation status. * = p < 0.05.

Article Snippet: Plus Reagent (Life Technologies, Carlsbad, CA, USA) or Turbofect (Thermo Fisher Scientific, Waltham, MA, USA) and p53 isoform expression plasmids (500 ng) were diluted in 100 μL of Opti-MEM medium (Life Technologies) with a ratio 1:1 (μL of Plus reagent: μg of transfected DNA).

Techniques: Gene Expression, Expressing, Amplification, Comparison, Mutagenesis

Journal: Cancers

Article Title: Altered Expression of Shorter p53 Family Isoforms Can Impact Melanoma Aggressiveness

doi: 10.3390/cancers13205231

Figure Lengend Snippet: The protein expression of p53/p73 isoforms in melanoma cell lines. ( A ) Western blot analysis of endogenous p53 expression in melanoma cell lines. Proteins from melanocytes (HEMa) and melanoma cell lines expressing wt p53 (WM793B, A375M, Mel501, A375, SK-Mel-24) or mut p53 (Mel-224, Mel-505, CHL1, WM983B, MeWo, SK-Mel-3, RPMI-7951), or that were devoid of p53 expression (LM6, ), were extracted as described. Fifty micrograms of protein extracts were analyzed by Western blot and p53 expression was revealed using either sheep polyclonal SAPU or KJC12 anti-human p53 antibodies. The anti-actin antibody was used as the loading control. ( B ) Western blot analysis of endogenous p73 expression in melanoma cell lines. Proteins from melanocytes (HEMa) and melanoma cell lines expressing wt (WM793B, A375M, Mel501, A375, SK-Mel-24) or mut p53 (Mel-224, Mel-505, CHL1, WM983B, MeWo, SK-Mel-3, RPMI-7951), or that were devoid of p53 expression (LM6), were extracted as described. Fifty micrograms of protein extracts were analyzed by Western blot. p73 expression was revealed using rabbit monoclonal EP436Y anti-p73 antibody. The anti-actin antibody was used as the loading control. Representative data of three independent experiments yielding similar results are shown.

Article Snippet: Plus Reagent (Life Technologies, Carlsbad, CA, USA) or Turbofect (Thermo Fisher Scientific, Waltham, MA, USA) and p53 isoform expression plasmids (500 ng) were diluted in 100 μL of Opti-MEM medium (Life Technologies) with a ratio 1:1 (μL of Plus reagent: μg of transfected DNA).

Techniques: Expressing, Western Blot, Control

Journal: Cancers

Article Title: Altered Expression of Shorter p53 Family Isoforms Can Impact Melanoma Aggressiveness

doi: 10.3390/cancers13205231

Figure Lengend Snippet: The expression of endogenous p53 isoforms in response to DNA-damaging agents commonly used in the clinic to treat cancer patients was evaluated in melanoma cell lines. Cells were treated with 1.5 µM doxorubicin ( A ), 10 µM dacarbazine ( B ), or 1 µM cisplatin or etoposide ( C ), and ( D ) γ-irradiated with 5 and 10 Gy (CTRL, non-irradiated cells; 0 h, cells collected immediately upon irradiation). Cells were harvested 24 h after the treatment ( A , B ), or at different time points after the treatment as indicated ( C , D ). α-actinin ( A , B ) and naphthol blue ( C , D ) were used as loading controls. To better identify the different p53 isoforms, transient transfections in the p53-null H1299 cells with single isoforms were included (arrows indicate the position on the blot of the intended p53 isoform, given that p53 isoforms can be post-translationally modified and multiple isoforms can be produced starting from downstream ATGs). ( A , B ) Respectively, 50 µg for the endogenous expression or 12.5 µg for the over-expression of p53 isoforms were loaded into the gels. ( C , D ) A total of 50 μg of proteins was analyzed by Western blot. Representative data of three independent experiments yielding similar results are shown.

Article Snippet: Plus Reagent (Life Technologies, Carlsbad, CA, USA) or Turbofect (Thermo Fisher Scientific, Waltham, MA, USA) and p53 isoform expression plasmids (500 ng) were diluted in 100 μL of Opti-MEM medium (Life Technologies) with a ratio 1:1 (μL of Plus reagent: μg of transfected DNA).

Techniques: Expressing, Irradiation, Transfection, Modification, Produced, Over Expression, Western Blot

Journal: Cancers

Article Title: Altered Expression of Shorter p53 Family Isoforms Can Impact Melanoma Aggressiveness

doi: 10.3390/cancers13205231

Figure Lengend Snippet: Localization and pro-oncogenic functions of Δ160p53 isoforms. ( A , B ) Immunofluorescence analysis of the subcellular localization of Δ160p53 proteins in H1299 cells stably over-expressing Δ160p53α, Δ160p53β, and Δ160p53γ isoforms, with the use of the p53 pantropic antibodies DO-12 (panel A ) and SAPU (panel B ). DAPI was used to counterstain the nuclei. ( C ) Cellular fractionation followed by Western blot of H1299 cells stably over-expressing Δ160p53 with or without 1.5 µM doxorubicin treatment for 24 h. SAPU antibody was used to detect the Δ160p53 isoforms. α-actinin was used as a loading control for the cytoplasmic protein fractions, whereas Histone H3 was used for the chromatin-bound protein fractions. ( D ) To measure the migration potential of H1299 stably over-expressing Δ160p53α, Δ160p53β, and Δ160p53γ, compared with the parental H1299 cells, we performed a wound-healing assay. Images were taken at different time points: 0, 8, and 24 h post scratch. Quantification of the migration potential after 24 h was obtained with the Wimasis Image Analysis tool. ( E ) The proliferation ability of H1299 clones stably over-expressing Δ160p53 isoforms compared with the empty control was evaluated with the colony formation assay. A total of 1000 cells was seeded into 6-well plates and left growing for 2 weeks. Visible clones were stained with crystal violet and manually counted. Bars in panels D and E represent the averages and the standard deviations of 3 biological experiments. ** = p < 0.01; * = p < 0.05.

Article Snippet: Plus Reagent (Life Technologies, Carlsbad, CA, USA) or Turbofect (Thermo Fisher Scientific, Waltham, MA, USA) and p53 isoform expression plasmids (500 ng) were diluted in 100 μL of Opti-MEM medium (Life Technologies) with a ratio 1:1 (μL of Plus reagent: μg of transfected DNA).

Techniques: Immunofluorescence, Stable Transfection, Expressing, Cell Fractionation, Western Blot, Control, Migration, Wound Healing Assay, Clone Assay, Colony Assay, Staining

Journal: Cancers

Article Title: Altered Expression of Shorter p53 Family Isoforms Can Impact Melanoma Aggressiveness

doi: 10.3390/cancers13205231

Figure Lengend Snippet: The expression of p53/p73 isoforms in parental and vemurafenib-resistant melanoma cell lines A375M-R and WM793B-R. ( A ) Expression of the TP53 gene in the vemurafenib-resistant melanoma cell lines. Relative expression levels of p53 isoforms analyzed by pre-amplification followed by qPCR. The expression was normalized to the expression level of total TP53 . ( B ) Western blot analysis of the endogenous p53 expression in parental and vemurafenib-resistant melanoma cell lines. p53 isoform expression was revealed using isoform-specific antibodies: TSRα and 421 to detect p53α isoforms; β-sheep and KJC8 to detect p53β isoforms. Naphthol blue staining or β-actin were used as loading controls. ( C ) Relative expression levels of TP73 isoforms were analyzed by qPCR. The expression was normalized to the geometric mean of GUSB and TBP. ( D ) Western blot analysis of p73 isoform expression in vemurafenib-resistant melanoma cell lines was revealed using pantropic rabbit polyclonal EP436Y anti-p73 antibody. β-actin was used as a loading control. ( A , C ) Average of two independently performed experiments ± SD is shown. One-way Anova with a post hoc Tukey–Kramer test was used. * = p -value < 0.05. ( B , D ) Representative data of three independent experiments yielding similar results are shown.

Article Snippet: Plus Reagent (Life Technologies, Carlsbad, CA, USA) or Turbofect (Thermo Fisher Scientific, Waltham, MA, USA) and p53 isoform expression plasmids (500 ng) were diluted in 100 μL of Opti-MEM medium (Life Technologies) with a ratio 1:1 (μL of Plus reagent: μg of transfected DNA).

Techniques: Expressing, Amplification, Western Blot, Staining, Control