p src y416  (Cell Signaling Technology Inc)

Journal: Molecular Medicine Reports

Article Title: The anticancer effects of cinobufagin on hepatocellular carcinoma Huh-7 cells are associated with activation of the p73 signaling pathway

doi: 10.3892/mmr.2019.10108

Figure Lengend Snippet: Cinobufagin may induce anticancer effects on Huh-7 cells via activation of p73 signaling. (A) Protein expression levels of p73, p-p73 (Y99), MDM2, p-MDM2 (S166), p21, Puma and Noxa in cells in cells following treatment with 5 µmol/l cinobufagin for 24 h, as determined by western blotting. Densitometric analysis of (B) p-p73, (C) Puma, (D) p-MDM2, (E) Noxa and (F) p21. (G) Expression of p73 in Huh-7 cells, as determined by immunocytochemistry. Representative images are shown at ×200 magnification. Data are presented as the means ± standard error of the mean of three independent experiments. *P<0.05 vs. control, # P<0.05 vs. cinobufagin. AURKA, aurora kinase A; MDM2, mouse double minute 2 homolog; Noxa, phorbol-12-myristate-13-acetate-induced protein 1; p, phosphorylated; Puma, p53 upregulated modulator of apoptosis.

Article Snippet: The rabbit monoclonal anti-AURKA (cat. no. 14475), rabbit polyclonal anti-p-p73 (Y99; cat. no. 4665) and anti-p-MDM2 (S166; cat. no. 3521) antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA).

Techniques: Activation Assay, Expressing, Western Blot, Immunocytochemistry, Control

Journal: Molecular Medicine Reports

Article Title: The anticancer effects of cinobufagin on hepatocellular carcinoma Huh-7 cells are associated with activation of the p73 signaling pathway

doi: 10.3892/mmr.2019.10108

Figure Lengend Snippet: Schematic diagram of cinobufagin-induced effects on hepatocellular carcinoma Huh-7 cells with mutant p53. Cinobufagin inhibits the viability, arrests the cell cycle and induces the apoptosis of Huh-7 cells by inhibiting AURKA and p53 signaling, and activating p73 signaling. AURKA, aurora kinase A; Bax, Bcl-2-associated X protein; Bcl-2, B-cell lymphoma 2; CDK1, cyclin-dependent kinase 1; hnRNPK, heterogeneous nuclear ribonucleoprotein K; NKA, Na + /K + -ATPase; Noxa, phorbol-12-myristate-13-acetate-induced protein 1; p, phosphorylated; PCNA, proliferating cell nuclear antigen; Puma, p53 upregulated modulator of apoptosis.

Article Snippet: The rabbit monoclonal anti-AURKA (cat. no. 14475), rabbit polyclonal anti-p-p73 (Y99; cat. no. 4665) and anti-p-MDM2 (S166; cat. no. 3521) antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA).

Techniques: Mutagenesis

Journal: Oncogene

Article Title: Restoring PUMA induction overcomes KRAS -mediated resistance to anti-EGFR antibodies in colorectal cancer

doi: 10.1038/s41388-018-0289-x

Figure Lengend Snippet: (A) DiFi cells transfected with control scrambled or p73 siRNA for 24 hr were re-plated and treated with 10 nM cetuximab (Cmab). Expression of p73 at 8 hr, and PUMA and cleaved (C) caspase-3 at 24 hr after cetuximab treatment was analyzed by western blotting. Cells without siRNA transfection and re-plating were used as the control for analyzing p73 at 8r after treatment. (B) Western blotting of indicated proteins in DiFi cells treated 10 nM cetuximab at the indicated time points. Phospho-p73 (p-p73, Y99); phospho-AKT (p-AKT, S473); phospho-ERK1/2 (p-ERK1/2, T202/Y204). (C) DiFi cells transfected with either a control empty vector or a HA-p73α construct were treated with 10 nM Cmab for the indicated times. Binding of transfected p73α to the PUMA promoter was analyzed by chromatin immunoprecipitation (ChIP) using anti-HA antibody with IgG as control, followed by PCR amplification and analysis of PCR products by agarose gel electrophoresis. (D) Crystal violet staining (upper panel) and MTS analysis (lower panel) of DiFi cells transfected with siRNA as in (A) and treated with Cmab at the indicated doses for 72 hr. (E) Western blotting of indicated proteins in DiFi cells transfected with control empty vector or constitutively active AKT for 6 hr, and then treated with 10 nM cetuximab for 8 or 24 hr. (F) Crystal violet staining (upper panel) and MTS analysis (lower panel) of DiFi cells transfected as in (E) and treated with Cmab at the indicated doses for 72 hr. (G) A model of PUMA induction by anti-EGFR antibodies.

Article Snippet: Western blotting was performed as previously described [ ] using antibodies against: β-Actin (A5441, Sigma), cleaved caspase-3 (#9661, Cell Signaling, Danvers, MA, USA), cleaved caspase-9 (#9502, Cell Signaling), Mcl-1 (#559027, BD Biosciences, San Jose, CA, USA), Bax (#610983, BD Biosciences), Bid (#2002, Cell Signaling), Bcl-xL (#610212, BD Biosciences), Bim (#2819, Cell Signaling), Bcl-2 (#M0887, Agilent DAKO, Santa Clara, CA, USA), cytochrome c (sc-7159, Santa Cruz Biotechnology, Santa Cruz, CA, USA), COX IV (A21348, Invitrogen), PUMA [ ], Bak (#06–536, EMD Millipore), Noxa (#OP180, EMD Millipore), p73 (A300–126A, Bethyl Laboratories, Montgomery, TX, USA), p-p73 (#4665, Cell Signaling), p-AKT (#4058, Cell Signaling), total AKT (#9272, Cell Signaling), p-ERK1/2 (#4376, Cell Signaling), total ERK1/2 (#9102, Cell Signaling), p-FoxO3A (#9464, Cell Signaling), total FoxO3A (07–702, EMD Millipore), p53 (sc-126, Santa Cruz), p-EGFR (#2234, Cell Signaling), total EGFR (#610016, BD Biosciences), KRAS (sc-30, Santa Cruz), p-Aurora A/B/C (#2914, Cell Signaling), total Aurora A (#4718, Cell Signaling), total Aurora B (#3094, Cell Signaling), and HA (#12CA5, Roche, Indianapolis, IN, USA).

Techniques: Transfection, Control, Expressing, Western Blot, Plasmid Preparation, Construct, Binding Assay, Chromatin Immunoprecipitation, Amplification, Agarose Gel Electrophoresis, Staining

Journal: Oncogene

Article Title: Restoring PUMA induction overcomes KRAS -mediated resistance to anti-EGFR antibodies in colorectal cancer

doi: 10.1038/s41388-018-0289-x

Figure Lengend Snippet: (A) MTS analysis of parental (red) and cetuximab-resistant (Cmab-R, black) DiFi cells treated with cetuximab (Cmab) or panitumumab (Pmab) at the indicated doses for 72 hr. (B) Western blotting of cleaved (C) caspase-3 in the parental and Cmab-R DiFi cells treated with 10 nM of Cmab or Pmab for 24 hr. (C) Western blotting of indicated proteins in the parental and Cmab-R DiFi cells. Lysates of Cmab-R DiFi cells were prepared from cells cultured in medium with 10 nM cetuximab (Cmab+), or without cetuximab (Cmab-) for 6 days. p-EGFR (Y1068); p-AKT (S473); p-ERK1/2 (T202/Y204). (D) Western blotting of indicated Bcl-2 family proteins in the parental and Cmab-R DiFi cells treated with 10 nM of Cmab for 24 hr. (E) Western blotting of phosphorylated (p-p73, Y99) and total p73 in the parental and Cmab-R DiFi cells treated with 10 nM Cmab for 8 hr. (F) Parental and Cmab-R DiFi cells transfected with control empty or HA-p73α-expressing vector were treated with 10 nM cetuximab for 8 hr. Binding of transfected p73α to the PUMA promoter was analyzed by chromatin immunoprecipitation (ChIP) using anti-HA antibody, followed by PCR amplification and analysis of PCR products by agarose gel electrophoresis. (G) Parental and Cmab-R DiFi cells were infected with EGFP-PUMA-expressing adenovirus (Ad-PUMA) at the indicated MOI for 24 hr. Upper, analysis of apoptosis by nuclear fragmentation; lower , western blotting of PUMA. Results were expressed as means ± s.e.m. of triplicates in two independent experiments. *** P <0.001.

Article Snippet: Western blotting was performed as previously described [ ] using antibodies against: β-Actin (A5441, Sigma), cleaved caspase-3 (#9661, Cell Signaling, Danvers, MA, USA), cleaved caspase-9 (#9502, Cell Signaling), Mcl-1 (#559027, BD Biosciences, San Jose, CA, USA), Bax (#610983, BD Biosciences), Bid (#2002, Cell Signaling), Bcl-xL (#610212, BD Biosciences), Bim (#2819, Cell Signaling), Bcl-2 (#M0887, Agilent DAKO, Santa Clara, CA, USA), cytochrome c (sc-7159, Santa Cruz Biotechnology, Santa Cruz, CA, USA), COX IV (A21348, Invitrogen), PUMA [ ], Bak (#06–536, EMD Millipore), Noxa (#OP180, EMD Millipore), p73 (A300–126A, Bethyl Laboratories, Montgomery, TX, USA), p-p73 (#4665, Cell Signaling), p-AKT (#4058, Cell Signaling), total AKT (#9272, Cell Signaling), p-ERK1/2 (#4376, Cell Signaling), total ERK1/2 (#9102, Cell Signaling), p-FoxO3A (#9464, Cell Signaling), total FoxO3A (07–702, EMD Millipore), p53 (sc-126, Santa Cruz), p-EGFR (#2234, Cell Signaling), total EGFR (#610016, BD Biosciences), KRAS (sc-30, Santa Cruz), p-Aurora A/B/C (#2914, Cell Signaling), total Aurora A (#4718, Cell Signaling), total Aurora B (#3094, Cell Signaling), and HA (#12CA5, Roche, Indianapolis, IN, USA).

Techniques: Western Blot, Cell Culture, Transfection, Control, Expressing, Plasmid Preparation, Binding Assay, Chromatin Immunoprecipitation, Amplification, Agarose Gel Electrophoresis, Infection

Journal: Oncogene

Article Title: Restoring PUMA induction overcomes KRAS -mediated resistance to anti-EGFR antibodies in colorectal cancer

doi: 10.1038/s41388-018-0289-x

Figure Lengend Snippet: (A) DiFi cells transfected with control scrambled or p73 siRNA for 24 hr were re-plated and treated with 10 nM cetuximab (Cmab). Expression of p73 at 8 hr, and PUMA and cleaved (C) caspase-3 at 24 hr after cetuximab treatment was analyzed by western blotting. Cells without siRNA transfection and re-plating were used as the control for analyzing p73 at 8r after treatment. (B) Western blotting of indicated proteins in DiFi cells treated 10 nM cetuximab at the indicated time points. Phospho-p73 (p-p73, Y99); phospho-AKT (p-AKT, S473); phospho-ERK1/2 (p-ERK1/2, T202/Y204). (C) DiFi cells transfected with either a control empty vector or a HA-p73α construct were treated with 10 nM Cmab for the indicated times. Binding of transfected p73α to the PUMA promoter was analyzed by chromatin immunoprecipitation (ChIP) using anti-HA antibody with IgG as control, followed by PCR amplification and analysis of PCR products by agarose gel electrophoresis. (D) Crystal violet staining (upper panel) and MTS analysis (lower panel) of DiFi cells transfected with siRNA as in (A) and treated with Cmab at the indicated doses for 72 hr. (E) Western blotting of indicated proteins in DiFi cells transfected with control empty vector or constitutively active AKT for 6 hr, and then treated with 10 nM cetuximab for 8 or 24 hr. (F) Crystal violet staining (upper panel) and MTS analysis (lower panel) of DiFi cells transfected as in (E) and treated with Cmab at the indicated doses for 72 hr. (G) A model of PUMA induction by anti-EGFR antibodies.

Article Snippet: Cell lysates were incubated with EZview Red Protein G Affinity Gel (Sigma) beads utilizing an antibody against p-p73 (#4665, Cell Signaling).

Techniques: Transfection, Control, Expressing, Western Blot, Plasmid Preparation, Construct, Binding Assay, Chromatin Immunoprecipitation, Amplification, Agarose Gel Electrophoresis, Staining

Journal: Oncogene

Article Title: Restoring PUMA induction overcomes KRAS -mediated resistance to anti-EGFR antibodies in colorectal cancer

doi: 10.1038/s41388-018-0289-x

Figure Lengend Snippet: (A) MTS analysis of parental (red) and cetuximab-resistant (Cmab-R, black) DiFi cells treated with cetuximab (Cmab) or panitumumab (Pmab) at the indicated doses for 72 hr. (B) Western blotting of cleaved (C) caspase-3 in the parental and Cmab-R DiFi cells treated with 10 nM of Cmab or Pmab for 24 hr. (C) Western blotting of indicated proteins in the parental and Cmab-R DiFi cells. Lysates of Cmab-R DiFi cells were prepared from cells cultured in medium with 10 nM cetuximab (Cmab+), or without cetuximab (Cmab-) for 6 days. p-EGFR (Y1068); p-AKT (S473); p-ERK1/2 (T202/Y204). (D) Western blotting of indicated Bcl-2 family proteins in the parental and Cmab-R DiFi cells treated with 10 nM of Cmab for 24 hr. (E) Western blotting of phosphorylated (p-p73, Y99) and total p73 in the parental and Cmab-R DiFi cells treated with 10 nM Cmab for 8 hr. (F) Parental and Cmab-R DiFi cells transfected with control empty or HA-p73α-expressing vector were treated with 10 nM cetuximab for 8 hr. Binding of transfected p73α to the PUMA promoter was analyzed by chromatin immunoprecipitation (ChIP) using anti-HA antibody, followed by PCR amplification and analysis of PCR products by agarose gel electrophoresis. (G) Parental and Cmab-R DiFi cells were infected with EGFP-PUMA-expressing adenovirus (Ad-PUMA) at the indicated MOI for 24 hr. Upper, analysis of apoptosis by nuclear fragmentation; lower , western blotting of PUMA. Results were expressed as means ± s.e.m. of triplicates in two independent experiments. *** P <0.001.

Article Snippet: Cell lysates were incubated with EZview Red Protein G Affinity Gel (Sigma) beads utilizing an antibody against p-p73 (#4665, Cell Signaling).

Techniques: Western Blot, Cell Culture, Transfection, Control, Expressing, Plasmid Preparation, Binding Assay, Chromatin Immunoprecipitation, Amplification, Agarose Gel Electrophoresis, Infection

Journal: Scientific Reports

Article Title: IER3 is a crucial mediator of TAp73β-induced apoptosis in cervical cancer and confers etoposide sensitivity

doi: 10.1038/srep08367

Figure Lengend Snippet: (A) HeLa cells were incubated with 0.1% dimethylsulfoxide (DMSO), etoposide (ETO, 200 μM), or doxorubicin (DOXO, 2 μM) for 24 h. Representative immunoblot (left panel) and quantitative analysis of the TAp73β and IER3 levels are shown (right panel). Symbols (* and # ) indicate significant values compared with the respective solvent controls, and the results represent three independent experiments run in triplicate ( p < 0.05). (B) The HeLa cells were transfected with scrambled or IER3-specific siRNAs, cells were treated with DMSO, ETO, or DOXO for 24 h, and their viability was measured. The HeLa cells were transfected with scrambled, p73-specific (C), or IER3-specific (D) siRNAs, they were incubated with increasing concentrations of ETO (left panel) or DOXO (right panel) for 24 h, and cell viability was measured. (E) HeLa cells were transfected with scrambled or p73-specific siRNA and then exposed to DMSO, ETO, or DOXO for 24 h. Using cell lysates, changes in the expression level of IER3 were determined by immunoblot analysis. Quantitative analysis of the IER3 levels is shown in the right panel (n = 3). (F) HeLa cells were transfected with the IER3-expressing plasmid and p73-specific siRNA as indicated and treated with DMSO or ETO, and their cellular viability was measured. All results are expressed as the mean ± SEM of three independent experiments performed in triplicate. Statistically significant values are indicated with different letters or asterisks ( p < 0.05). (A and E) These full-blots membrane was cut into pieces according to estimated molecular weight of proteins of interest and probed with indicated antibodies. All cropped bolts have been run under the same experimental condition.

Article Snippet: The anti-Caspase 3 (9662), 8 (9746), 9 (9508), anti-FLAG (2368) and anti-p-p73 (4665) antibodies were purchased from Cell Signaling (Danvers, MA, USA).

Techniques: Incubation, Western Blot, Solvent, Transfection, Expressing, Plasmid Preparation, Membrane, Molecular Weight

Journal: Scientific Reports

Article Title: IER3 is a crucial mediator of TAp73β-induced apoptosis in cervical cancer and confers etoposide sensitivity

doi: 10.1038/srep08367

Figure Lengend Snippet: (A) Equal amounts of extracted protein from FFPE tissues of normal human cervical epithelium (n = 9) and cervical carcinoma tissues (n = 10) were subjected to SDS-PAGE for immunoblot analysis using indicated antibodies (upper panel). The full-blots membrane was cut into pieces according to estimated molecular weight of proteins of interest and probed with indicated antibodies. All cropped bolts have been run under the same experimental condition. Areas of cervical tissues scrapped for protein extraction are indicated with dotted lines (lower panel). The relative quantified expression of both p73β and IER3 proteins between the control cervix and cervical cancer were compared. Estimated regression line superimposed on scatter plot of levels of p73β and IER3 proteins in normal cervix is also presented with a correlation coefficient. (B) The total mRNAs from FFPE tissues of control human cervix (n = 5) and cervical carcinoma (n = 5) were extracted and used for a quantitative real-time PCR analysis of p73 and IER3 . (C) Representative immunohistochemistical analyses of p73 and IER3 expression in control cervix and cervical cancer are shown.

Article Snippet: The anti-Caspase 3 (9662), 8 (9746), 9 (9508), anti-FLAG (2368) and anti-p-p73 (4665) antibodies were purchased from Cell Signaling (Danvers, MA, USA).

Techniques: SDS Page, Western Blot, Membrane, Molecular Weight, Protein Extraction, Expressing, Control, Real-time Polymerase Chain Reaction