p53 1c12 mouse mab  (Cell Signaling Technology Inc)

 
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 90
    Name:
    p53 1C12 Mouse mAb
    Description:
    The p53 tumor suppressor protein plays a major role in cellular response to DNA damage and other genomic aberrations Activation of p53 can lead to either cell cycle arrest and DNA repair or apoptosis 1 p53 is phosphorylated at multiple sites in vivo and by several different protein kinases in vitro 2 3 DNA damage induces phosphorylation of p53 at Ser15 and Ser20 and leads to a reduced interaction between p53 and its negative regulator the oncoprotein MDM2 4 MDM2 inhibits p53 accumulation by targeting it for ubiquitination and proteasomal degradation 5 6 p53 can be phosphorylated by ATM ATR and DNA PK at Ser15 and Ser37 Phosphorylation impairs the ability of MDM2 to bind p53 promoting both the accumulation and activation of p53 in response to DNA damage 4 7 Chk2 and Chk1 can phosphorylate p53 at Ser20 enhancing its tetramerization stability and activity 8 9 p53 is phosphorylated at Ser392 in vivo 10 11 and by CAK in vitro 11 Phosphorylation of p53 at Ser392 is increased in human tumors 12 and has been reported to influence the growth suppressor function DNA binding and transcriptional activation of p53 10 13 14 p53 is phosphorylated at Ser6 and Ser9 by CK1δ and CK1ε both in vitro and in vivo 13 15 Phosphorylation of p53 at Ser46 regulates the ability of p53 to induce apoptosis 16 Acetylation of p53 is mediated by p300 and CBP acetyltransferases Inhibition of deacetylation suppressing MDM2 from recruiting HDAC1 complex by p19 ARF stabilizes p53 Acetylation appears to play a positive role in the accumulation of p53 protein in stress response 17 Following DNA damage human p53 becomes acetylated at Lys382 Lys379 in mouse in vivo to enhance p53 DNA binding 18 Deacetylation of p53 occurs through interaction with the SIRT1 protein a deacetylase that may be involved in cellular aging and the DNA damage response 19
    Catalog Number:
    2524
    Price:
    None
    Applications:
    Western Blot, Immunoprecipitation, Immunofluorescence, Flow Cytometry, Chromatin Immunoprecipitation
    Category:
    Primary Antibodies
    Source:
    Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Ser20 of human p53.
    Reactivity:
    Human Mouse Rat Hamster Monkey
    Buy from Supplier


    Structured Review

    Cell Signaling Technology Inc p53 1c12 mouse mab
    The p53 tumor suppressor protein plays a major role in cellular response to DNA damage and other genomic aberrations Activation of p53 can lead to either cell cycle arrest and DNA repair or apoptosis 1 p53 is phosphorylated at multiple sites in vivo and by several different protein kinases in vitro 2 3 DNA damage induces phosphorylation of p53 at Ser15 and Ser20 and leads to a reduced interaction between p53 and its negative regulator the oncoprotein MDM2 4 MDM2 inhibits p53 accumulation by targeting it for ubiquitination and proteasomal degradation 5 6 p53 can be phosphorylated by ATM ATR and DNA PK at Ser15 and Ser37 Phosphorylation impairs the ability of MDM2 to bind p53 promoting both the accumulation and activation of p53 in response to DNA damage 4 7 Chk2 and Chk1 can phosphorylate p53 at Ser20 enhancing its tetramerization stability and activity 8 9 p53 is phosphorylated at Ser392 in vivo 10 11 and by CAK in vitro 11 Phosphorylation of p53 at Ser392 is increased in human tumors 12 and has been reported to influence the growth suppressor function DNA binding and transcriptional activation of p53 10 13 14 p53 is phosphorylated at Ser6 and Ser9 by CK1δ and CK1ε both in vitro and in vivo 13 15 Phosphorylation of p53 at Ser46 regulates the ability of p53 to induce apoptosis 16 Acetylation of p53 is mediated by p300 and CBP acetyltransferases Inhibition of deacetylation suppressing MDM2 from recruiting HDAC1 complex by p19 ARF stabilizes p53 Acetylation appears to play a positive role in the accumulation of p53 protein in stress response 17 Following DNA damage human p53 becomes acetylated at Lys382 Lys379 in mouse in vivo to enhance p53 DNA binding 18 Deacetylation of p53 occurs through interaction with the SIRT1 protein a deacetylase that may be involved in cellular aging and the DNA damage response 19
    https://www.bioz.com/result/p53 1c12 mouse mab/product/Cell Signaling Technology Inc
    Average 90 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    p53 1c12 mouse mab - by Bioz Stars, 2020-02
    90/100 stars

    Images

    Related Articles

    Immunocytochemistry:

    Article Title: Mitochondrial Translocation of p53 Modulates Neuronal Fate by Preventing Differentiation-Induced Mitochondrial Stress
    Article Snippet: Paragraph title: Immunocytochemistry and confocal microscopy ... Cells were incubated with a primary mouse monoclonal antibody reactive to p53 (2524; Cell Signaling Technology, Inc.) at a dilution of 1:1000 or with a primary rabbit polyclonal antibody reactive to LC3 (PA1-16931; Thermo Fisher Scientific, Inc.) at a dilution of 1:200, overnight at 4°C.

    Blocking Assay:

    Article Title: Synuclein gamma expression enhances radiation resistance of breast cancer cells
    Article Snippet: Membranes were blocked at room temperature in a blocking buffer (phosphate-buffered saline (PBS) containing 0.2% of casein (VWR E666) and 0.05% Tween 20) and probed with indicated primary antibodies overnight at 4°C. .. Primary antibodies were SNCG (Abcam, ab55424, 1:1000), β-actin (Santa-Cruz, sc-47778, 1:200), phosphor-p53 (Ser15) (Cell Signaling, #9284, 1:1000), p53 (Cell Signaling, #2524, 1:1000), p21 Waf1/Cip1 (Cell Signaling, #2947, 1:1000), Bax (Santa-Cruz, sc-7480, 1:1000) and Bcl-xL (Cell Signaling, #2764, 1:1000).

    Electrophoresis:

    Article Title: Tauroursodeoxycholic acid increases neural stem cell pool and neuronal conversion by regulating mitochondria-cell cycle retrograde signaling
    Article Snippet: .. Briefly, 50–80 μg of protein extracts were separated on 12 or 15% sodium dodecyl sulfate-polyacrylamide electrophoresis gels (SDS-PAGE) and then subjected to immunoblotting using primary mouse monoclonal antibodies reactive to cytochrome c (556433; BD Biosciences PharMingen), p53 (2524; Cell Signaling Technology®, Inc.), p27 (sc-1641; Santa Cruz Biotechnology, Inc.) and Sox2 (MAB2018; R & D Systems® Inc.); primary rabbit polyclonal antibodies reactive to p21 (sc-397-G; Santa Cruz Biotechnology, Inc.). .. Blots were subsequently incubated with secondary antibodies conjugated with anti-mouse and anti-rabbit IgG conjugated with horseradish peroxidase (Bio-Rad Laboratories) and anti-goat IgG-HRP (sc-2020; Santa Cruz Biotechnology, Inc.) for 2 h at room temperature.

    Article Title: Analysis of the relationship between the KRAS G12V oncogene and the Hippo effector YAP1 in embryonal rhabdomyosarcoma
    Article Snippet: Whole cell lysate were separated via SDS-PAGE electrophoresis and transferred to 0.2 μM PVDF Western blotting membrane. .. Membranes were then probed with mouse anti-Yap/Taz (Santa Cruz, 101199, 1:100), rabbit anti-phospho Yap Ser127 (Cell Signaling, #9411, 1:1000) (this antibody can detect murine pYap S112 and pTaz S89 ), pan-Ras (BD Bioscience, #610001, 1:1000), p53 (Cell Signaling, #2524, 1:1000), p16-INK4A (Proteintech, #10883-1-AP, 1:500), small-T-antigen (Millipore, #D01, 1:1000), tubulin (Sigma, #B512, 1:2000), actin (Sigma, #A5060, 1:2000) and Gapdh (Abcam, #Ab8245, 1:10000).

    Article Title: Mitochondrial Translocation of p53 Modulates Neuronal Fate by Preventing Differentiation-Induced Mitochondrial Stress
    Article Snippet: .. Briefly, 20–80 μg of total, mitochondrial, and cytosolic proteins were separated on 8%, 12%, or 15% sodium dodecyl sulfate–polyacrylamide electrophoresis gels and then subjected to immunoblotting using primary mouse monoclonal antibodies reactive to p53 (2524; Cell Signaling Technology® , Inc., Boston, MA) and cytochrome c (556433; BD Biosciences Pharmingen, San Diego, CA); primary rabbit polyclonal antibodies reactive to LC3 (PA1-16931; Thermo Fisher Scientific, Inc.), Parkin (sc-30130; Santa Cruz Biotechnology, Inc., Dallas, TX), and MnSOD (sc-30080; Santa Cruz Biotechnology, Inc.); or primary goat polyclonal antibodies reactive to mouse RECQL4 (sc-16927; Santa Cruz Biotechnology, Inc.) and human RECQL4 (sc-16924; Santa Cruz Biotechnology, Inc.). .. Blots were subsequently incubated with secondary antibodies conjugated with anti-mouse and anti-rabbit immunoglobulin G (IgG) conjugated with horseradish peroxidase (Bio-Rad Laboratories) and anti-goat IgG-HRP (sc-2020; Santa Cruz Biotechnology, Inc.) for 2 h at room temperature.

    Incubation:

    Article Title: Mitochondrial Translocation of p53 Modulates Neuronal Fate by Preventing Differentiation-Induced Mitochondrial Stress
    Article Snippet: .. Cells were incubated with a primary mouse monoclonal antibody reactive to p53 (2524; Cell Signaling Technology, Inc.) at a dilution of 1:1000 or with a primary rabbit polyclonal antibody reactive to LC3 (PA1-16931; Thermo Fisher Scientific, Inc.) at a dilution of 1:200, overnight at 4°C. .. After two washes, the secondary DyLight 488-conjugated anti-mouse antibody (Jackson ImmunoResearch Laboratories, Inc.) diluted 1:100 or the secondary Alexa Fluor 488 (A-21206; Invitrogen, Life Technologies Corp.) diluted 1:200, was added to cells for 2 h at room temperature.

    Article Title: Tauroursodeoxycholic acid increases neural stem cell pool and neuronal conversion by regulating mitochondria-cell cycle retrograde signaling
    Article Snippet: Briefly, 50–80 μg of protein extracts were separated on 12 or 15% sodium dodecyl sulfate-polyacrylamide electrophoresis gels (SDS-PAGE) and then subjected to immunoblotting using primary mouse monoclonal antibodies reactive to cytochrome c (556433; BD Biosciences PharMingen), p53 (2524; Cell Signaling Technology®, Inc.), p27 (sc-1641; Santa Cruz Biotechnology, Inc.) and Sox2 (MAB2018; R & D Systems® Inc.); primary rabbit polyclonal antibodies reactive to p21 (sc-397-G; Santa Cruz Biotechnology, Inc.). .. Blots were subsequently incubated with secondary antibodies conjugated with anti-mouse and anti-rabbit IgG conjugated with horseradish peroxidase (Bio-Rad Laboratories) and anti-goat IgG-HRP (sc-2020; Santa Cruz Biotechnology, Inc.) for 2 h at room temperature.

    Article Title: An essential role for Ink4 and Cip/Kip cell-cycle inhibitors in preventing replicative stress
    Article Snippet: Cells were permeabilized with 0.25% BSA, 0.5% Triton X-100 in PBS for 10 min and blocked with washing buffer 3% BSA for 1 h. Neurospheres or C17.2 cells were incubated with specific antibodies against Sox9 (1 : 250, Millipore, AB5535), phospho-RPA (1 : 100; S4/S8; Bethyl Laboratories, Montgomery, TX, USA), 53BP1 (1 : 100; ab36823; Abcam, Cambridge, UK), phospho-p53-Ser15 (1 : 100; Cell Signaling, 9284 S), p21Cip1 (1 : 100, sc-6246, Santa Cruz Biotechnology, Santa Cruz, CA, USA), p27Kip1 (1 : 100; BD Biosciences, 610241) and γ H2AX (1 : 750, Millipore, 05-636), and secondary fluorescence antibodies (Alexa-594 goat anti-mouse and Alexa-647 goat anti-Rabbit; 1 : 250, Invitrogen). .. The following primary antibodies were used for detection of proteins in these immunoblots: cyclin D1 (1 : 500; clone M-2, Santa Cruz Biotechnology, sc-718), Cyclin A2 (1:500; H-432, Santa Cruz Biotechnology, sc-751), Rb phospho-Ser 780 (1 : 250; Cell Signaling, 9307), Rb (1 : 250; G3-245, BD Biosciences, 554136), alpha-tubulin (1:2000; Sigma, T 9026), vinculin (1:2500; Sigma, V9131), Cdk4 (1 : 1000; Abcam, ab137675), cyclin E (1 : 1000; Abcam, ab7959-1), Cdk6 (a gift from M Barbacid), p21Cip1 (1 : 250, sc-397, Santa Cruz Biotechnology), p27Kip1 (1 : 1000; BD Biosciences, 610241), p53 (1 : 1000; Cell Signaling Technology, 2524).

    Article Title: Mitochondrial Translocation of p53 Modulates Neuronal Fate by Preventing Differentiation-Induced Mitochondrial Stress
    Article Snippet: Briefly, 20–80 μg of total, mitochondrial, and cytosolic proteins were separated on 8%, 12%, or 15% sodium dodecyl sulfate–polyacrylamide electrophoresis gels and then subjected to immunoblotting using primary mouse monoclonal antibodies reactive to p53 (2524; Cell Signaling Technology® , Inc., Boston, MA) and cytochrome c (556433; BD Biosciences Pharmingen, San Diego, CA); primary rabbit polyclonal antibodies reactive to LC3 (PA1-16931; Thermo Fisher Scientific, Inc.), Parkin (sc-30130; Santa Cruz Biotechnology, Inc., Dallas, TX), and MnSOD (sc-30080; Santa Cruz Biotechnology, Inc.); or primary goat polyclonal antibodies reactive to mouse RECQL4 (sc-16927; Santa Cruz Biotechnology, Inc.) and human RECQL4 (sc-16924; Santa Cruz Biotechnology, Inc.). .. Blots were subsequently incubated with secondary antibodies conjugated with anti-mouse and anti-rabbit immunoglobulin G (IgG) conjugated with horseradish peroxidase (Bio-Rad Laboratories) and anti-goat IgG-HRP (sc-2020; Santa Cruz Biotechnology, Inc.) for 2 h at room temperature.

    Article Title: Synuclein gamma expression enhances radiation resistance of breast cancer cells
    Article Snippet: Membranes were washed with PBS-0.05% Tween 20 for 30 min and incubated with secondary antibodies conjugated with HorseRadish Peroxydase (HRP) for 1 hour. .. Primary antibodies were SNCG (Abcam, ab55424, 1:1000), β-actin (Santa-Cruz, sc-47778, 1:200), phosphor-p53 (Ser15) (Cell Signaling, #9284, 1:1000), p53 (Cell Signaling, #2524, 1:1000), p21 Waf1/Cip1 (Cell Signaling, #2947, 1:1000), Bax (Santa-Cruz, sc-7480, 1:1000) and Bcl-xL (Cell Signaling, #2764, 1:1000).

    Article Title: Reversible p53 inhibition prevents cisplatin ototoxicity without blocking chemotherapeutic efficacy
    Article Snippet: .. Blots were incubated with mouse monoclonal antibodies against phospho‐Chk1 (1/1,000, Ser345, Cell Signaling Technology, #2348), p53 (1/1,500, Cell Signaling Technology, #2524), and β‐actin (1/10,000, Sigma‐Aldrich, #A1978), and with polyclonal rabbit antibodies against phospho‐H2AX (1/1500, Ser139, Cell Signaling Technology, #2577), phospho‐p53 (1/1500, Ser15, Cell Signaling Technology, #9289), and cleaved caspase‐3 (1/1,000, Asp175, Cell Signaling Technology, #9661), prior to incubation with horseradish peroxidase‐conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, #115‐035‐144 and 111‐035‐144). ..

    Expressing:

    Article Title: Reversible p53 inhibition prevents cisplatin ototoxicity without blocking chemotherapeutic efficacy
    Article Snippet: Paragraph title: Expression levels of DNA damage response proteins and apoptosis ... Blots were incubated with mouse monoclonal antibodies against phospho‐Chk1 (1/1,000, Ser345, Cell Signaling Technology, #2348), p53 (1/1,500, Cell Signaling Technology, #2524), and β‐actin (1/10,000, Sigma‐Aldrich, #A1978), and with polyclonal rabbit antibodies against phospho‐H2AX (1/1500, Ser139, Cell Signaling Technology, #2577), phospho‐p53 (1/1500, Ser15, Cell Signaling Technology, #9289), and cleaved caspase‐3 (1/1,000, Asp175, Cell Signaling Technology, #9661), prior to incubation with horseradish peroxidase‐conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, #115‐035‐144 and 111‐035‐144).

    BIA-KA:

    Article Title: P53 and mTOR signalling determine fitness selection through cell competition during early mouse embryonic development
    Article Snippet: Western blotting Cells lysates were collected using Laemmli buffer (0.05 M Tris-HCl at pH 6.8, 1% SDS, 10% glycerol, 0.1% β-mercaptoethanol), quantified using BCA quantification (Thermo Fisher Scientific) and resolved using Criterion XT pre-cast gels (BioRad) followed by transfer to PVDF membranes. .. Antibodies used are as follows: pS6S240/244 #5264, S6 #2317, p53 #2524, p4E-BP1T37/46 #2855, total 4E-BP1 #9644, cleaved caspase-3 #9664, cleaved PARP #9544, pAKTT473 #4060, TSC2 #4308, β-actin #4970; all from Cell Signaling Technology and used at 1:1000 dilutions.

    Article Title: An essential role for Ink4 and Cip/Kip cell-cycle inhibitors in preventing replicative stress
    Article Snippet: The following primary antibodies were used for detection of proteins in these immunoblots: cyclin D1 (1 : 500; clone M-2, Santa Cruz Biotechnology, sc-718), Cyclin A2 (1:500; H-432, Santa Cruz Biotechnology, sc-751), Rb phospho-Ser 780 (1 : 250; Cell Signaling, 9307), Rb (1 : 250; G3-245, BD Biosciences, 554136), alpha-tubulin (1:2000; Sigma, T 9026), vinculin (1:2500; Sigma, V9131), Cdk4 (1 : 1000; Abcam, ab137675), cyclin E (1 : 1000; Abcam, ab7959-1), Cdk6 (a gift from M Barbacid), p21Cip1 (1 : 250, sc-397, Santa Cruz Biotechnology), p27Kip1 (1 : 1000; BD Biosciences, 610241), p53 (1 : 1000; Cell Signaling Technology, 2524). .. The following primary antibodies were used for detection of proteins in these immunoblots: cyclin D1 (1 : 500; clone M-2, Santa Cruz Biotechnology, sc-718), Cyclin A2 (1:500; H-432, Santa Cruz Biotechnology, sc-751), Rb phospho-Ser 780 (1 : 250; Cell Signaling, 9307), Rb (1 : 250; G3-245, BD Biosciences, 554136), alpha-tubulin (1:2000; Sigma, T 9026), vinculin (1:2500; Sigma, V9131), Cdk4 (1 : 1000; Abcam, ab137675), cyclin E (1 : 1000; Abcam, ab7959-1), Cdk6 (a gift from M Barbacid), p21Cip1 (1 : 250, sc-397, Santa Cruz Biotechnology), p27Kip1 (1 : 1000; BD Biosciences, 610241), p53 (1 : 1000; Cell Signaling Technology, 2524).

    Article Title: Synuclein gamma expression enhances radiation resistance of breast cancer cells
    Article Snippet: The protein concentrations of the supernatant were quantified by bicinchoninic acid (BCA) protein assay kit (Pierce® , ThermoFisher). .. Primary antibodies were SNCG (Abcam, ab55424, 1:1000), β-actin (Santa-Cruz, sc-47778, 1:200), phosphor-p53 (Ser15) (Cell Signaling, #9284, 1:1000), p53 (Cell Signaling, #2524, 1:1000), p21 Waf1/Cip1 (Cell Signaling, #2947, 1:1000), Bax (Santa-Cruz, sc-7480, 1:1000) and Bcl-xL (Cell Signaling, #2764, 1:1000).

    Modification:

    Article Title: Analysis of the relationship between the KRAS G12V oncogene and the Hippo effector YAP1 in embryonal rhabdomyosarcoma
    Article Snippet: Cells were lysed in modified RIPA buffer supplemented with protease and phosphatase cocktail inhibitors (Sigma). .. Membranes were then probed with mouse anti-Yap/Taz (Santa Cruz, 101199, 1:100), rabbit anti-phospho Yap Ser127 (Cell Signaling, #9411, 1:1000) (this antibody can detect murine pYap S112 and pTaz S89 ), pan-Ras (BD Bioscience, #610001, 1:1000), p53 (Cell Signaling, #2524, 1:1000), p16-INK4A (Proteintech, #10883-1-AP, 1:500), small-T-antigen (Millipore, #D01, 1:1000), tubulin (Sigma, #B512, 1:2000), actin (Sigma, #A5060, 1:2000) and Gapdh (Abcam, #Ab8245, 1:10000).

    Western Blot:

    Article Title: Tauroursodeoxycholic acid increases neural stem cell pool and neuronal conversion by regulating mitochondria-cell cycle retrograde signaling
    Article Snippet: Levels of cytochrome c, p53, p21, p27 and Sox2 were determined by Western blot analysis of total, mitochondrial and cytosolic protein extracts, obtained as previously described. .. Briefly, 50–80 μg of protein extracts were separated on 12 or 15% sodium dodecyl sulfate-polyacrylamide electrophoresis gels (SDS-PAGE) and then subjected to immunoblotting using primary mouse monoclonal antibodies reactive to cytochrome c (556433; BD Biosciences PharMingen), p53 (2524; Cell Signaling Technology®, Inc.), p27 (sc-1641; Santa Cruz Biotechnology, Inc.) and Sox2 (MAB2018; R & D Systems® Inc.); primary rabbit polyclonal antibodies reactive to p21 (sc-397-G; Santa Cruz Biotechnology, Inc.).

    Article Title: Analysis of the relationship between the KRAS G12V oncogene and the Hippo effector YAP1 in embryonal rhabdomyosarcoma
    Article Snippet: Paragraph title: Western blotting ... Membranes were then probed with mouse anti-Yap/Taz (Santa Cruz, 101199, 1:100), rabbit anti-phospho Yap Ser127 (Cell Signaling, #9411, 1:1000) (this antibody can detect murine pYap S112 and pTaz S89 ), pan-Ras (BD Bioscience, #610001, 1:1000), p53 (Cell Signaling, #2524, 1:1000), p16-INK4A (Proteintech, #10883-1-AP, 1:500), small-T-antigen (Millipore, #D01, 1:1000), tubulin (Sigma, #B512, 1:2000), actin (Sigma, #A5060, 1:2000) and Gapdh (Abcam, #Ab8245, 1:10000).

    Article Title: 1,25‐Dihydroxyvitamin D exerts an antiaging role by activation of Nrf2‐antioxidant signaling and inactivation of p16/p53‐senescence signaling, et al. 1,25‐Dihydroxyvitamin D exerts an antiaging role by activation of Nrf2‐antioxidant signaling and inactivation of p16/p53‐senescence signaling
    Article Snippet: Paragraph title: Western blot ... Primary antibodies against peroxiredoxin‐I (PrdxI) (sc‐7381; Santa Cruz Biotechnology), Bmi1 (05‐637; Millipore), p16 (sc‐377412; Santa Cruz Biotechnology), p53 (#2524; Cell Signaling Technology), p21 (sc‐471; Santa Cruz Biotechnology), Wnt16 (sc‐20964, Santa Cruz Biotechnology), caspase‐3 (#9662; Cell signaling technology), cyclin D1 (#2922; Cell signaling technology), cyclin E (sc‐377100; Santa Cruz Biotechnology), and β‐actin (AP0060; Bioworld Technology) were used.

    Article Title: P53 and mTOR signalling determine fitness selection through cell competition during early mouse embryonic development
    Article Snippet: Paragraph title: Western blotting ... Antibodies used are as follows: pS6S240/244 #5264, S6 #2317, p53 #2524, p4E-BP1T37/46 #2855, total 4E-BP1 #9644, cleaved caspase-3 #9664, cleaved PARP #9544, pAKTT473 #4060, TSC2 #4308, β-actin #4970; all from Cell Signaling Technology and used at 1:1000 dilutions.

    Article Title: A lincRNA-p21/miR-181 family feedback loop regulates microglial activation during systemic LPS- and MPTP- induced neuroinflammation
    Article Snippet: Paragraph title: Western blotting ... Membranes were then blocked with 5% non-fat dry milk in TBS with 0.1% Tween and probed with appropriate primary antibodies against PKC-δ (1:1,000, Santa Cruz Biotech, sc-9379), iNOS (1:1000, Santa Cruz Biotech, sc-650), p53 (1:1000, Cell Signaling, #2524), Ago2 (1:1000, Abcam, ab32381), histone H3 (1:1000, Abcam, ab4729), and GAPDH (1:5000, Bioworld, AP0063) overnight at 4 °C.

    Article Title: Tonic Activation of Bax Primes Neural Progenitors for Rapid Apoptosis through a Mechanism Preserved in Medulloblastoma
    Article Snippet: Paragraph title: Western blot analysis. ... Immunologic analysis was performed on a SNAP ID device (Millipore) using manufacturer's protocol with primary antibodies to β-actin (Cell Signaling Technology, cat#4970), Bax-N20 (Santa Cruz Biotechnology, sc-493), Bcl-XL (Cell Signaling Technology, cat#2764), Bad (Cell Signaling Technology, cat#9239), Bim (Cell Signaling Technology, cat#2933), Puma (Cell Signaling Technology, cat#7467), cleaved caspase-3 (cC3; Cell Signaling Technology, cat#9664), Cyclin D2 (Cell Signaling Technology, #3741), Cox4 (Clontech, Cat. No. 630105), and p53 (Cell Signaling Technology, cat#2524), Mcl-1 (Cell Signaling Technology, cat#5453), Bcl-2 (Cell Signaling Technology, cat#3498), and phospho-ERK1/2 (Cell Signaling Technology, cat#4370).

    Article Title: An essential role for Ink4 and Cip/Kip cell-cycle inhibitors in preventing replicative stress
    Article Snippet: .. The following primary antibodies were used for detection of proteins in these immunoblots: cyclin D1 (1 : 500; clone M-2, Santa Cruz Biotechnology, sc-718), Cyclin A2 (1:500; H-432, Santa Cruz Biotechnology, sc-751), Rb phospho-Ser 780 (1 : 250; Cell Signaling, 9307), Rb (1 : 250; G3-245, BD Biosciences, 554136), alpha-tubulin (1:2000; Sigma, T 9026), vinculin (1:2500; Sigma, V9131), Cdk4 (1 : 1000; Abcam, ab137675), cyclin E (1 : 1000; Abcam, ab7959-1), Cdk6 (a gift from M Barbacid), p21Cip1 (1 : 250, sc-397, Santa Cruz Biotechnology), p27Kip1 (1 : 1000; BD Biosciences, 610241), p53 (1 : 1000; Cell Signaling Technology, 2524). .. Horseradish peroxidase-conjugated goat anti-mouse and anti-rabbit immunoglobulins (Dako) were used as secondary antibodies.

    Article Title: Mitochondrial Translocation of p53 Modulates Neuronal Fate by Preventing Differentiation-Induced Mitochondrial Stress
    Article Snippet: Levels of p53, cytochrome c , LC3, RECQL4, Parkin, and MnSOD were determined by western blot analysis. .. Briefly, 20–80 μg of total, mitochondrial, and cytosolic proteins were separated on 8%, 12%, or 15% sodium dodecyl sulfate–polyacrylamide electrophoresis gels and then subjected to immunoblotting using primary mouse monoclonal antibodies reactive to p53 (2524; Cell Signaling Technology® , Inc., Boston, MA) and cytochrome c (556433; BD Biosciences Pharmingen, San Diego, CA); primary rabbit polyclonal antibodies reactive to LC3 (PA1-16931; Thermo Fisher Scientific, Inc.), Parkin (sc-30130; Santa Cruz Biotechnology, Inc., Dallas, TX), and MnSOD (sc-30080; Santa Cruz Biotechnology, Inc.); or primary goat polyclonal antibodies reactive to mouse RECQL4 (sc-16927; Santa Cruz Biotechnology, Inc.) and human RECQL4 (sc-16924; Santa Cruz Biotechnology, Inc.).

    Article Title: Synuclein gamma expression enhances radiation resistance of breast cancer cells
    Article Snippet: Paragraph title: Western blot analysis and antibodies ... Primary antibodies were SNCG (Abcam, ab55424, 1:1000), β-actin (Santa-Cruz, sc-47778, 1:200), phosphor-p53 (Ser15) (Cell Signaling, #9284, 1:1000), p53 (Cell Signaling, #2524, 1:1000), p21 Waf1/Cip1 (Cell Signaling, #2947, 1:1000), Bax (Santa-Cruz, sc-7480, 1:1000) and Bcl-xL (Cell Signaling, #2764, 1:1000).

    Cell Harvesting:

    Article Title: Mitochondrial Translocation of p53 Modulates Neuronal Fate by Preventing Differentiation-Induced Mitochondrial Stress
    Article Snippet: For visualization of p53 mitochondrial and LC3-II mitochondrial colocalizations in NSCs, cells were incubated with 0.5 μ M MitoTracker® Red CMXRos (M-7512; Molecular Probes, Life Technologies Corp.), which preferentially accumulates in mitochondria, for 30 min at 37°C before cell harvesting. .. Cells were incubated with a primary mouse monoclonal antibody reactive to p53 (2524; Cell Signaling Technology, Inc.) at a dilution of 1:1000 or with a primary rabbit polyclonal antibody reactive to LC3 (PA1-16931; Thermo Fisher Scientific, Inc.) at a dilution of 1:200, overnight at 4°C.

    Cell Culture:

    Article Title: Tonic Activation of Bax Primes Neural Progenitors for Rapid Apoptosis through a Mechanism Preserved in Medulloblastoma
    Article Snippet: Cultured cells, whole cerebella, and tumors were lysed by homogenization in lysis buffer (Cell Signaling Technology, cat#9803). .. Immunologic analysis was performed on a SNAP ID device (Millipore) using manufacturer's protocol with primary antibodies to β-actin (Cell Signaling Technology, cat#4970), Bax-N20 (Santa Cruz Biotechnology, sc-493), Bcl-XL (Cell Signaling Technology, cat#2764), Bad (Cell Signaling Technology, cat#9239), Bim (Cell Signaling Technology, cat#2933), Puma (Cell Signaling Technology, cat#7467), cleaved caspase-3 (cC3; Cell Signaling Technology, cat#9664), Cyclin D2 (Cell Signaling Technology, #3741), Cox4 (Clontech, Cat. No. 630105), and p53 (Cell Signaling Technology, cat#2524), Mcl-1 (Cell Signaling Technology, cat#5453), Bcl-2 (Cell Signaling Technology, cat#3498), and phospho-ERK1/2 (Cell Signaling Technology, cat#4370).

    other:

    Article Title: Epigallocatechin-3-gallate, a prototypic chemopreventative agent for protection against cisplatin-based ototoxicity
    Article Snippet: Antibodies Various primary antibodies (with catalog numbers and companies) used were as follows: p-ERK1/2 (no. 7976), ERK1/2 (no. 93), STAT1 (no. 592), Na+/K+ ATPase α 1 (no. 21712) STAT3 (no. 8019), Bcl-xL (no. 8392), β -actin (no. 69879) from Santa Cruz Biotechnology (Dallas, TX, USA); JAK2 (no. 3230), pJAK2 (no. 3776), p-STAT3 Tyr705 (no. 4113 S), p-STAT1 Ser727 (no. 9177 S), p53 (no. 2524), Bcl-2 (no. 2876), cleaved caspase-3 (no. 9661), Bax (no. 2772), TNFα (no. 3707 S), Cox2 (no. 4842) from Cell Signaling Technology (Danvers, MA, USA); myosin VIIa rabbit polyclonal (no. 25-6790) from Proteus Biosciences (Ramona, CA, USA); CtBP2 mouse IgG1 (no. 612044) from BD Biosciences (San Jose, CA, USA); Alexa Fluor 488 Phalloidin (no. A-12379) from Thermo Fisher Scientific (Waltham, MA, USA).

    Imaging:

    Article Title: Analysis of the relationship between the KRAS G12V oncogene and the Hippo effector YAP1 in embryonal rhabdomyosarcoma
    Article Snippet: Membranes were then probed with mouse anti-Yap/Taz (Santa Cruz, 101199, 1:100), rabbit anti-phospho Yap Ser127 (Cell Signaling, #9411, 1:1000) (this antibody can detect murine pYap S112 and pTaz S89 ), pan-Ras (BD Bioscience, #610001, 1:1000), p53 (Cell Signaling, #2524, 1:1000), p16-INK4A (Proteintech, #10883-1-AP, 1:500), small-T-antigen (Millipore, #D01, 1:1000), tubulin (Sigma, #B512, 1:2000), actin (Sigma, #A5060, 1:2000) and Gapdh (Abcam, #Ab8245, 1:10000). .. Primary antibody binding was then visualised using species-specific conjugated secondary antibodies (Invitrogen) and digital imaging.

    Article Title: Synuclein gamma expression enhances radiation resistance of breast cancer cells
    Article Snippet: Protein-antibody complexes were visualized by chemoluminescence with the SuperSignal® West Dura Extended Duration Substrate (Thermo scientific), using a LAS-3000 imaging system (Fujifilm, Tokyo, Japan) or X-ray films (CL-Xposure TM Film, Thermo scientific). .. Primary antibodies were SNCG (Abcam, ab55424, 1:1000), β-actin (Santa-Cruz, sc-47778, 1:200), phosphor-p53 (Ser15) (Cell Signaling, #9284, 1:1000), p53 (Cell Signaling, #2524, 1:1000), p21 Waf1/Cip1 (Cell Signaling, #2947, 1:1000), Bax (Santa-Cruz, sc-7480, 1:1000) and Bcl-xL (Cell Signaling, #2764, 1:1000).

    Protein Concentration:

    Article Title: An essential role for Ink4 and Cip/Kip cell-cycle inhibitors in preventing replicative stress
    Article Snippet: The following primary antibodies were used for detection of proteins in these immunoblots: cyclin D1 (1 : 500; clone M-2, Santa Cruz Biotechnology, sc-718), Cyclin A2 (1:500; H-432, Santa Cruz Biotechnology, sc-751), Rb phospho-Ser 780 (1 : 250; Cell Signaling, 9307), Rb (1 : 250; G3-245, BD Biosciences, 554136), alpha-tubulin (1:2000; Sigma, T 9026), vinculin (1:2500; Sigma, V9131), Cdk4 (1 : 1000; Abcam, ab137675), cyclin E (1 : 1000; Abcam, ab7959-1), Cdk6 (a gift from M Barbacid), p21Cip1 (1 : 250, sc-397, Santa Cruz Biotechnology), p27Kip1 (1 : 1000; BD Biosciences, 610241), p53 (1 : 1000; Cell Signaling Technology, 2524). .. The following primary antibodies were used for detection of proteins in these immunoblots: cyclin D1 (1 : 500; clone M-2, Santa Cruz Biotechnology, sc-718), Cyclin A2 (1:500; H-432, Santa Cruz Biotechnology, sc-751), Rb phospho-Ser 780 (1 : 250; Cell Signaling, 9307), Rb (1 : 250; G3-245, BD Biosciences, 554136), alpha-tubulin (1:2000; Sigma, T 9026), vinculin (1:2500; Sigma, V9131), Cdk4 (1 : 1000; Abcam, ab137675), cyclin E (1 : 1000; Abcam, ab7959-1), Cdk6 (a gift from M Barbacid), p21Cip1 (1 : 250, sc-397, Santa Cruz Biotechnology), p27Kip1 (1 : 1000; BD Biosciences, 610241), p53 (1 : 1000; Cell Signaling Technology, 2524).

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Reversible p53 inhibition prevents cisplatin ototoxicity without blocking chemotherapeutic efficacy
    Article Snippet: Blots were incubated with mouse monoclonal antibodies against phospho‐Chk1 (1/1,000, Ser345, Cell Signaling Technology, #2348), p53 (1/1,500, Cell Signaling Technology, #2524), and β‐actin (1/10,000, Sigma‐Aldrich, #A1978), and with polyclonal rabbit antibodies against phospho‐H2AX (1/1500, Ser139, Cell Signaling Technology, #2577), phospho‐p53 (1/1500, Ser15, Cell Signaling Technology, #9289), and cleaved caspase‐3 (1/1,000, Asp175, Cell Signaling Technology, #9661), prior to incubation with horseradish peroxidase‐conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, #115‐035‐144 and 111‐035‐144). .. As a specific antibody against ATR was not commercially available for cochlear tissue analysis, we used PCR to evaluate the expression of ATR and its substrate, Chk1. mRNA was extracted using a RiboPure kit (Ambion). cDNA synthesis and conventional PCR were performed using the Long Range 2Step RT–PCR kit (Qiagen), according to the manufacturer's instructions.

    Binding Assay:

    Article Title: Analysis of the relationship between the KRAS G12V oncogene and the Hippo effector YAP1 in embryonal rhabdomyosarcoma
    Article Snippet: Membranes were then probed with mouse anti-Yap/Taz (Santa Cruz, 101199, 1:100), rabbit anti-phospho Yap Ser127 (Cell Signaling, #9411, 1:1000) (this antibody can detect murine pYap S112 and pTaz S89 ), pan-Ras (BD Bioscience, #610001, 1:1000), p53 (Cell Signaling, #2524, 1:1000), p16-INK4A (Proteintech, #10883-1-AP, 1:500), small-T-antigen (Millipore, #D01, 1:1000), tubulin (Sigma, #B512, 1:2000), actin (Sigma, #A5060, 1:2000) and Gapdh (Abcam, #Ab8245, 1:10000). .. Primary antibody binding was then visualised using species-specific conjugated secondary antibodies (Invitrogen) and digital imaging.

    MTT Assay:

    Article Title: SP600125 has a remarkable anticancer potential against undifferentiated thyroid cancer through selective action on ROCK and p53 pathways
    Article Snippet: .. SP600125 (1,9-Pyrazoloanthrone), MTT (3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2-tetrazolium-bromide), Monoclonal anti-β-Catenin antibody 6F9, Monoclonal Anti-α-Tubulin clone DM1A, Monoclonal Anti-Acetylated Tubulin Clone 6-11B-1 were purchased from Sigma-Aldrich. p53 antibody, Phospho-p53 (Ser46) antibody, p53 (1C12) Mouse mAb, p21 Waf1/Cip1 (DCS60) Mouse mAb, p21 Waf1/Cip1 (12D1) Rabbit mAb, Acetyl-p53 (Lys382) antibody, Phospho-p53 (Ser15), antibody, Phospho-Histone H2A.X 20E3 antibody, HDAC6 D2E5 antibody and Acetyl- β-Catenin (Lys49) antibody were purchased from Cell Signaling Technologies. .. ProLong Gold Antifade Reagent with DAPI, Wheat Germ Agglutinin Alexa-Fluor 594 Conjugate, Alexa-fluor conjugated and HRP conjugated secondary antibodies were purchased from Life Technologies.

    Fluorescence:

    Article Title: An essential role for Ink4 and Cip/Kip cell-cycle inhibitors in preventing replicative stress
    Article Snippet: Cells were permeabilized with 0.25% BSA, 0.5% Triton X-100 in PBS for 10 min and blocked with washing buffer 3% BSA for 1 h. Neurospheres or C17.2 cells were incubated with specific antibodies against Sox9 (1 : 250, Millipore, AB5535), phospho-RPA (1 : 100; S4/S8; Bethyl Laboratories, Montgomery, TX, USA), 53BP1 (1 : 100; ab36823; Abcam, Cambridge, UK), phospho-p53-Ser15 (1 : 100; Cell Signaling, 9284 S), p21Cip1 (1 : 100, sc-6246, Santa Cruz Biotechnology, Santa Cruz, CA, USA), p27Kip1 (1 : 100; BD Biosciences, 610241) and γ H2AX (1 : 750, Millipore, 05-636), and secondary fluorescence antibodies (Alexa-594 goat anti-mouse and Alexa-647 goat anti-Rabbit; 1 : 250, Invitrogen). .. The following primary antibodies were used for detection of proteins in these immunoblots: cyclin D1 (1 : 500; clone M-2, Santa Cruz Biotechnology, sc-718), Cyclin A2 (1:500; H-432, Santa Cruz Biotechnology, sc-751), Rb phospho-Ser 780 (1 : 250; Cell Signaling, 9307), Rb (1 : 250; G3-245, BD Biosciences, 554136), alpha-tubulin (1:2000; Sigma, T 9026), vinculin (1:2500; Sigma, V9131), Cdk4 (1 : 1000; Abcam, ab137675), cyclin E (1 : 1000; Abcam, ab7959-1), Cdk6 (a gift from M Barbacid), p21Cip1 (1 : 250, sc-397, Santa Cruz Biotechnology), p27Kip1 (1 : 1000; BD Biosciences, 610241), p53 (1 : 1000; Cell Signaling Technology, 2524).

    Immunodetection:

    Article Title: An essential role for Ink4 and Cip/Kip cell-cycle inhibitors in preventing replicative stress
    Article Snippet: Paragraph title: Immunodetection of proteins ... The following primary antibodies were used for detection of proteins in these immunoblots: cyclin D1 (1 : 500; clone M-2, Santa Cruz Biotechnology, sc-718), Cyclin A2 (1:500; H-432, Santa Cruz Biotechnology, sc-751), Rb phospho-Ser 780 (1 : 250; Cell Signaling, 9307), Rb (1 : 250; G3-245, BD Biosciences, 554136), alpha-tubulin (1:2000; Sigma, T 9026), vinculin (1:2500; Sigma, V9131), Cdk4 (1 : 1000; Abcam, ab137675), cyclin E (1 : 1000; Abcam, ab7959-1), Cdk6 (a gift from M Barbacid), p21Cip1 (1 : 250, sc-397, Santa Cruz Biotechnology), p27Kip1 (1 : 1000; BD Biosciences, 610241), p53 (1 : 1000; Cell Signaling Technology, 2524).

    Microscopy:

    Article Title: An essential role for Ink4 and Cip/Kip cell-cycle inhibitors in preventing replicative stress
    Article Snippet: Cells were finally mounted in prolong anti-fade with DAPI (Invitrogen) and visualized using a confocal microscope SP2 (Leica, Wetzlar, Germany). .. The following primary antibodies were used for detection of proteins in these immunoblots: cyclin D1 (1 : 500; clone M-2, Santa Cruz Biotechnology, sc-718), Cyclin A2 (1:500; H-432, Santa Cruz Biotechnology, sc-751), Rb phospho-Ser 780 (1 : 250; Cell Signaling, 9307), Rb (1 : 250; G3-245, BD Biosciences, 554136), alpha-tubulin (1:2000; Sigma, T 9026), vinculin (1:2500; Sigma, V9131), Cdk4 (1 : 1000; Abcam, ab137675), cyclin E (1 : 1000; Abcam, ab7959-1), Cdk6 (a gift from M Barbacid), p21Cip1 (1 : 250, sc-397, Santa Cruz Biotechnology), p27Kip1 (1 : 1000; BD Biosciences, 610241), p53 (1 : 1000; Cell Signaling Technology, 2524).

    Purification:

    Article Title: SP600125 has a remarkable anticancer potential against undifferentiated thyroid cancer through selective action on ROCK and p53 pathways
    Article Snippet: SP600125 (1,9-Pyrazoloanthrone), MTT (3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2-tetrazolium-bromide), Monoclonal anti-β-Catenin antibody 6F9, Monoclonal Anti-α-Tubulin clone DM1A, Monoclonal Anti-Acetylated Tubulin Clone 6-11B-1 were purchased from Sigma-Aldrich. p53 antibody, Phospho-p53 (Ser46) antibody, p53 (1C12) Mouse mAb, p21 Waf1/Cip1 (DCS60) Mouse mAb, p21 Waf1/Cip1 (12D1) Rabbit mAb, Acetyl-p53 (Lys382) antibody, Phospho-p53 (Ser15), antibody, Phospho-Histone H2A.X 20E3 antibody, HDAC6 D2E5 antibody and Acetyl- β-Catenin (Lys49) antibody were purchased from Cell Signaling Technologies. .. Purified Mouse Anti-Actin Ab-5 was purchased from BD Biosciences.

    Polymerase Chain Reaction:

    Article Title: Reversible p53 inhibition prevents cisplatin ototoxicity without blocking chemotherapeutic efficacy
    Article Snippet: Blots were incubated with mouse monoclonal antibodies against phospho‐Chk1 (1/1,000, Ser345, Cell Signaling Technology, #2348), p53 (1/1,500, Cell Signaling Technology, #2524), and β‐actin (1/10,000, Sigma‐Aldrich, #A1978), and with polyclonal rabbit antibodies against phospho‐H2AX (1/1500, Ser139, Cell Signaling Technology, #2577), phospho‐p53 (1/1500, Ser15, Cell Signaling Technology, #9289), and cleaved caspase‐3 (1/1,000, Asp175, Cell Signaling Technology, #9661), prior to incubation with horseradish peroxidase‐conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, #115‐035‐144 and 111‐035‐144). .. As a specific antibody against ATR was not commercially available for cochlear tissue analysis, we used PCR to evaluate the expression of ATR and its substrate, Chk1. mRNA was extracted using a RiboPure kit (Ambion). cDNA synthesis and conventional PCR were performed using the Long Range 2Step RT–PCR kit (Qiagen), according to the manufacturer's instructions.

    Protein Extraction:

    Article Title: Dynamic Bcl-xL (S49) and (S62) Phosphorylation/Dephosphorylation during Mitosis Prevents Chromosome Instability and Aneuploidy in Normal Human Diploid Fibroblasts
    Article Snippet: Paragraph title: Protein extraction and immunoblotting ... The antibodies (Abs) in this study were Bcl-xL (54H6) rabbit monoclonal Ab (mAb), Ki-67(8D5) mouse mAb, p21Waf1/Cip1(12D1) rabbit mAb, p16/INK4A rabbit polyclonal Ab (pAb) and p53(1C12) mouse mAb obtained from Cell Signaling Technology Inc. (Beverly, MA).

    Confocal Microscopy:

    Article Title: Mitochondrial Translocation of p53 Modulates Neuronal Fate by Preventing Differentiation-Induced Mitochondrial Stress
    Article Snippet: Paragraph title: Immunocytochemistry and confocal microscopy ... Cells were incubated with a primary mouse monoclonal antibody reactive to p53 (2524; Cell Signaling Technology, Inc.) at a dilution of 1:1000 or with a primary rabbit polyclonal antibody reactive to LC3 (PA1-16931; Thermo Fisher Scientific, Inc.) at a dilution of 1:200, overnight at 4°C.

    Chloramphenicol Acetyltransferase Assay:

    Article Title: Tonic Activation of Bax Primes Neural Progenitors for Rapid Apoptosis through a Mechanism Preserved in Medulloblastoma
    Article Snippet: .. Immunologic analysis was performed on a SNAP ID device (Millipore) using manufacturer's protocol with primary antibodies to β-actin (Cell Signaling Technology, cat4970), Bax-N20 (Santa Cruz Biotechnology, sc-493), Bcl-XL (Cell Signaling Technology, cat#2764), Bad (Cell Signaling Technology, cat#9239), Bim (Cell Signaling Technology, cat#2933), Puma (Cell Signaling Technology, cat#7467), cleaved caspase-3 (cC3; Cell Signaling Technology, cat#9664), Cyclin D2 (Cell Signaling Technology, #3741), Cox4 (Clontech, Cat. No. 630105), and p53 (Cell Signaling Technology, cat#2524), Mcl-1 (Cell Signaling Technology, cat#5453), Bcl-2 (Cell Signaling Technology, cat#3498), and phospho-ERK1/2 (Cell Signaling Technology, cat#4370). .. Secondary antibodies were anti-rabbit IgG HRP (Cell Signaling Technology, cat#7074) and anti-mouse IgG HRP (Cell Signaling Technology, cat#7076).

    SDS Page:

    Article Title: Tauroursodeoxycholic acid increases neural stem cell pool and neuronal conversion by regulating mitochondria-cell cycle retrograde signaling
    Article Snippet: .. Briefly, 50–80 μg of protein extracts were separated on 12 or 15% sodium dodecyl sulfate-polyacrylamide electrophoresis gels (SDS-PAGE) and then subjected to immunoblotting using primary mouse monoclonal antibodies reactive to cytochrome c (556433; BD Biosciences PharMingen), p53 (2524; Cell Signaling Technology®, Inc.), p27 (sc-1641; Santa Cruz Biotechnology, Inc.) and Sox2 (MAB2018; R & D Systems® Inc.); primary rabbit polyclonal antibodies reactive to p21 (sc-397-G; Santa Cruz Biotechnology, Inc.). .. Blots were subsequently incubated with secondary antibodies conjugated with anti-mouse and anti-rabbit IgG conjugated with horseradish peroxidase (Bio-Rad Laboratories) and anti-goat IgG-HRP (sc-2020; Santa Cruz Biotechnology, Inc.) for 2 h at room temperature.

    Article Title: Analysis of the relationship between the KRAS G12V oncogene and the Hippo effector YAP1 in embryonal rhabdomyosarcoma
    Article Snippet: Whole cell lysate were separated via SDS-PAGE electrophoresis and transferred to 0.2 μM PVDF Western blotting membrane. .. Membranes were then probed with mouse anti-Yap/Taz (Santa Cruz, 101199, 1:100), rabbit anti-phospho Yap Ser127 (Cell Signaling, #9411, 1:1000) (this antibody can detect murine pYap S112 and pTaz S89 ), pan-Ras (BD Bioscience, #610001, 1:1000), p53 (Cell Signaling, #2524, 1:1000), p16-INK4A (Proteintech, #10883-1-AP, 1:500), small-T-antigen (Millipore, #D01, 1:1000), tubulin (Sigma, #B512, 1:2000), actin (Sigma, #A5060, 1:2000) and Gapdh (Abcam, #Ab8245, 1:10000).

    Article Title: A lincRNA-p21/miR-181 family feedback loop regulates microglial activation during systemic LPS- and MPTP- induced neuroinflammation
    Article Snippet: Briefly, equal amounts of the proteins were loaded on 10% or 15% SDS-PAGE gels for separation under reducing conditions, followed by blotting of the protein on a PVDF membrane (Immobilon-P, Millipore) in a Mini Trans-Blot electrophoretic transfer cell (Bio-Rad). .. Membranes were then blocked with 5% non-fat dry milk in TBS with 0.1% Tween and probed with appropriate primary antibodies against PKC-δ (1:1,000, Santa Cruz Biotech, sc-9379), iNOS (1:1000, Santa Cruz Biotech, sc-650), p53 (1:1000, Cell Signaling, #2524), Ago2 (1:1000, Abcam, ab32381), histone H3 (1:1000, Abcam, ab4729), and GAPDH (1:5000, Bioworld, AP0063) overnight at 4 °C.

    Software:

    Article Title: A lincRNA-p21/miR-181 family feedback loop regulates microglial activation during systemic LPS- and MPTP- induced neuroinflammation
    Article Snippet: Membranes were then blocked with 5% non-fat dry milk in TBS with 0.1% Tween and probed with appropriate primary antibodies against PKC-δ (1:1,000, Santa Cruz Biotech, sc-9379), iNOS (1:1000, Santa Cruz Biotech, sc-650), p53 (1:1000, Cell Signaling, #2524), Ago2 (1:1000, Abcam, ab32381), histone H3 (1:1000, Abcam, ab4729), and GAPDH (1:5000, Bioworld, AP0063) overnight at 4 °C. .. HRP-conjugated secondary antibodies (goat anti mouse/rabbit IgG 1:5000) were used for antibody detection with the immobilon ECL kit (Merck Millipore) in a ChemiDoc XRS + system (Bio-Rad) and band intensities were quantified using ImageJ software (US National Institutes of Health) .

    Homogenization:

    Article Title: Tonic Activation of Bax Primes Neural Progenitors for Rapid Apoptosis through a Mechanism Preserved in Medulloblastoma
    Article Snippet: Cultured cells, whole cerebella, and tumors were lysed by homogenization in lysis buffer (Cell Signaling Technology, cat#9803). .. Immunologic analysis was performed on a SNAP ID device (Millipore) using manufacturer's protocol with primary antibodies to β-actin (Cell Signaling Technology, cat#4970), Bax-N20 (Santa Cruz Biotechnology, sc-493), Bcl-XL (Cell Signaling Technology, cat#2764), Bad (Cell Signaling Technology, cat#9239), Bim (Cell Signaling Technology, cat#2933), Puma (Cell Signaling Technology, cat#7467), cleaved caspase-3 (cC3; Cell Signaling Technology, cat#9664), Cyclin D2 (Cell Signaling Technology, #3741), Cox4 (Clontech, Cat. No. 630105), and p53 (Cell Signaling Technology, cat#2524), Mcl-1 (Cell Signaling Technology, cat#5453), Bcl-2 (Cell Signaling Technology, cat#3498), and phospho-ERK1/2 (Cell Signaling Technology, cat#4370).

    Fractionation:

    Article Title: Tauroursodeoxycholic acid increases neural stem cell pool and neuronal conversion by regulating mitochondria-cell cycle retrograde signaling
    Article Snippet: Briefly, 50–80 μg of protein extracts were separated on 12 or 15% sodium dodecyl sulfate-polyacrylamide electrophoresis gels (SDS-PAGE) and then subjected to immunoblotting using primary mouse monoclonal antibodies reactive to cytochrome c (556433; BD Biosciences PharMingen), p53 (2524; Cell Signaling Technology®, Inc.), p27 (sc-1641; Santa Cruz Biotechnology, Inc.) and Sox2 (MAB2018; R & D Systems® Inc.); primary rabbit polyclonal antibodies reactive to p21 (sc-397-G; Santa Cruz Biotechnology, Inc.). .. VDAC (4866; Cell Signaling Technology®, Inc.), GAPDH (sc-32233; Santa Cruz Biotechnology, Inc.), β-actin (A5441; Sigma-Aldrich Corp.) and Lamin B1 (ab16048; Abcam®) antibodies were used as loading controls and/or to monitor the purity of mitochondrial and cytosolic fractionation.

    Article Title: Mitochondrial Translocation of p53 Modulates Neuronal Fate by Preventing Differentiation-Induced Mitochondrial Stress
    Article Snippet: Briefly, 20–80 μg of total, mitochondrial, and cytosolic proteins were separated on 8%, 12%, or 15% sodium dodecyl sulfate–polyacrylamide electrophoresis gels and then subjected to immunoblotting using primary mouse monoclonal antibodies reactive to p53 (2524; Cell Signaling Technology® , Inc., Boston, MA) and cytochrome c (556433; BD Biosciences Pharmingen, San Diego, CA); primary rabbit polyclonal antibodies reactive to LC3 (PA1-16931; Thermo Fisher Scientific, Inc.), Parkin (sc-30130; Santa Cruz Biotechnology, Inc., Dallas, TX), and MnSOD (sc-30080; Santa Cruz Biotechnology, Inc.); or primary goat polyclonal antibodies reactive to mouse RECQL4 (sc-16927; Santa Cruz Biotechnology, Inc.) and human RECQL4 (sc-16924; Santa Cruz Biotechnology, Inc.). .. VDAC (4866; Cell Signaling Technology, Inc.), GAPDH (sc-32233; Santa Cruz Biotechnology, Inc.), β-actin (A5441; Sigma-Aldrich Corp.), and Lamin B1 (ab16048; Abcam® , Cambridge, United Kingdom) antibodies were used as loading controls and/or to monitor the purity of mitochondrial and cytosolic fractionation.

    Lysis:

    Article Title: Tonic Activation of Bax Primes Neural Progenitors for Rapid Apoptosis through a Mechanism Preserved in Medulloblastoma
    Article Snippet: Cultured cells, whole cerebella, and tumors were lysed by homogenization in lysis buffer (Cell Signaling Technology, cat#9803). .. Immunologic analysis was performed on a SNAP ID device (Millipore) using manufacturer's protocol with primary antibodies to β-actin (Cell Signaling Technology, cat#4970), Bax-N20 (Santa Cruz Biotechnology, sc-493), Bcl-XL (Cell Signaling Technology, cat#2764), Bad (Cell Signaling Technology, cat#9239), Bim (Cell Signaling Technology, cat#2933), Puma (Cell Signaling Technology, cat#7467), cleaved caspase-3 (cC3; Cell Signaling Technology, cat#9664), Cyclin D2 (Cell Signaling Technology, #3741), Cox4 (Clontech, Cat. No. 630105), and p53 (Cell Signaling Technology, cat#2524), Mcl-1 (Cell Signaling Technology, cat#5453), Bcl-2 (Cell Signaling Technology, cat#3498), and phospho-ERK1/2 (Cell Signaling Technology, cat#4370).

    Article Title: Dynamic Bcl-xL (S49) and (S62) Phosphorylation/Dephosphorylation during Mitosis Prevents Chromosome Instability and Aneuploidy in Normal Human Diploid Fibroblasts
    Article Snippet: Protein extraction and immunoblotting To prepare total protein, cells were extracted with lysis buffer containing 20 mM Hepes- KOH, pH 7.4, 120 mM NaCl, 1% Triton X-100, 2 mM phenylmethylsulfonyl fluoride, a cocktail of protease inhibitors (CompleteTM , Roche Applied Science, Laval QC) and a cocktail of phosphatase inhibitors (PhosStopTM , Roche Applied Science). .. The antibodies (Abs) in this study were Bcl-xL (54H6) rabbit monoclonal Ab (mAb), Ki-67(8D5) mouse mAb, p21Waf1/Cip1(12D1) rabbit mAb, p16/INK4A rabbit polyclonal Ab (pAb) and p53(1C12) mouse mAb obtained from Cell Signaling Technology Inc. (Beverly, MA).

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 90
    Cell Signaling Technology Inc p53
    Effect of ATM or <t>p53</t> inhibition on DNA damage repair and hair cell survival 3D images of OHC nuclei taken from the basal regions of organ of Corti cultures treated with medium alone, 10 μM KU55933, 10 μM CDDP, or 10 μM CDDP in combination with 10 μM KU55933 for 1 day and immunolabeled for 53BP1 (green) and Hoechst 33342 (blue). Scale bar = 5 μm. Quantification analysis of 53BP1 foci number per nucleus in both IHCs (red bars) and OHCs (blue bars) for all conditions ( n = 50 nuclei per condition and per time point). Data are expressed as mean ± SEM. One‐way ANOVA followed by post hoc Tukey's test (*** P ≤ 0.0004; CDDP versus control, or CDDP versus CDDP + KU55933). Confocal images showing the basal region of the organ of Corti cultures treated with medium alone, 10 μM KU55933, 10 μM CDDP, or 10 μM CDDP in combination with 10 μM KU55933 for 5 days and immunolabeled for myosin 7A (red). Scale bar = 24 μm. Histograms representing the numbers of surviving IHCs (red bars) and OHCs (blue bars) for all treatment conditions after 5 days ( n = 5 cochleae per condition and per time point). Data are expressed as mean ± SEM. One‐way ANOVA test followed by post hoc Tukey's test (* P = 0.03, ** P = 0.004, *** P ≤ 0.0008; CDDP versus control, or CDDP versus CDDP + KU55933). 3D images of OHC nuclei from the basal regions of organ of Corti cultures treated with culture medium alone, 100 μM PFT‐α, 10 μM CDDP, or 10 μM CDDP in combination with 100 μM PFT‐α for 1 day and immunolabeled for 53BP1 (green) and Hoechst 33342 (blue). Scale bar = 5 μm. Quantification of 53BP1 foci number per nucleus in both IHCs (red bars) and OHCs (blue bars) for all conditions ( n = 50 nuclei per condition and per time point). Data are expressed as mean ± SEM. One‐way ANOVA test followed by post hoc Tukey's test (*** P ≤ 0.0007; CDDP versus control). Data information: All experiments were performed in triplicate.
    P53, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 90/100, based on 73 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/p53/product/Cell Signaling Technology Inc
    Average 90 stars, based on 73 article reviews
    Price from $9.99 to $1999.99
    p53 - by Bioz Stars, 2020-02
    90/100 stars
      Buy from Supplier

    Image Search Results


    Effect of ATM or p53 inhibition on DNA damage repair and hair cell survival 3D images of OHC nuclei taken from the basal regions of organ of Corti cultures treated with medium alone, 10 μM KU55933, 10 μM CDDP, or 10 μM CDDP in combination with 10 μM KU55933 for 1 day and immunolabeled for 53BP1 (green) and Hoechst 33342 (blue). Scale bar = 5 μm. Quantification analysis of 53BP1 foci number per nucleus in both IHCs (red bars) and OHCs (blue bars) for all conditions ( n = 50 nuclei per condition and per time point). Data are expressed as mean ± SEM. One‐way ANOVA followed by post hoc Tukey's test (*** P ≤ 0.0004; CDDP versus control, or CDDP versus CDDP + KU55933). Confocal images showing the basal region of the organ of Corti cultures treated with medium alone, 10 μM KU55933, 10 μM CDDP, or 10 μM CDDP in combination with 10 μM KU55933 for 5 days and immunolabeled for myosin 7A (red). Scale bar = 24 μm. Histograms representing the numbers of surviving IHCs (red bars) and OHCs (blue bars) for all treatment conditions after 5 days ( n = 5 cochleae per condition and per time point). Data are expressed as mean ± SEM. One‐way ANOVA test followed by post hoc Tukey's test (* P = 0.03, ** P = 0.004, *** P ≤ 0.0008; CDDP versus control, or CDDP versus CDDP + KU55933). 3D images of OHC nuclei from the basal regions of organ of Corti cultures treated with culture medium alone, 100 μM PFT‐α, 10 μM CDDP, or 10 μM CDDP in combination with 100 μM PFT‐α for 1 day and immunolabeled for 53BP1 (green) and Hoechst 33342 (blue). Scale bar = 5 μm. Quantification of 53BP1 foci number per nucleus in both IHCs (red bars) and OHCs (blue bars) for all conditions ( n = 50 nuclei per condition and per time point). Data are expressed as mean ± SEM. One‐way ANOVA test followed by post hoc Tukey's test (*** P ≤ 0.0007; CDDP versus control). Data information: All experiments were performed in triplicate.

    Journal: EMBO Molecular Medicine

    Article Title: Reversible p53 inhibition prevents cisplatin ototoxicity without blocking chemotherapeutic efficacy

    doi: 10.15252/emmm.201606230

    Figure Lengend Snippet: Effect of ATM or p53 inhibition on DNA damage repair and hair cell survival 3D images of OHC nuclei taken from the basal regions of organ of Corti cultures treated with medium alone, 10 μM KU55933, 10 μM CDDP, or 10 μM CDDP in combination with 10 μM KU55933 for 1 day and immunolabeled for 53BP1 (green) and Hoechst 33342 (blue). Scale bar = 5 μm. Quantification analysis of 53BP1 foci number per nucleus in both IHCs (red bars) and OHCs (blue bars) for all conditions ( n = 50 nuclei per condition and per time point). Data are expressed as mean ± SEM. One‐way ANOVA followed by post hoc Tukey's test (*** P ≤ 0.0004; CDDP versus control, or CDDP versus CDDP + KU55933). Confocal images showing the basal region of the organ of Corti cultures treated with medium alone, 10 μM KU55933, 10 μM CDDP, or 10 μM CDDP in combination with 10 μM KU55933 for 5 days and immunolabeled for myosin 7A (red). Scale bar = 24 μm. Histograms representing the numbers of surviving IHCs (red bars) and OHCs (blue bars) for all treatment conditions after 5 days ( n = 5 cochleae per condition and per time point). Data are expressed as mean ± SEM. One‐way ANOVA test followed by post hoc Tukey's test (* P = 0.03, ** P = 0.004, *** P ≤ 0.0008; CDDP versus control, or CDDP versus CDDP + KU55933). 3D images of OHC nuclei from the basal regions of organ of Corti cultures treated with culture medium alone, 100 μM PFT‐α, 10 μM CDDP, or 10 μM CDDP in combination with 100 μM PFT‐α for 1 day and immunolabeled for 53BP1 (green) and Hoechst 33342 (blue). Scale bar = 5 μm. Quantification of 53BP1 foci number per nucleus in both IHCs (red bars) and OHCs (blue bars) for all conditions ( n = 50 nuclei per condition and per time point). Data are expressed as mean ± SEM. One‐way ANOVA test followed by post hoc Tukey's test (*** P ≤ 0.0007; CDDP versus control). Data information: All experiments were performed in triplicate.

    Article Snippet: Blots were incubated with mouse monoclonal antibodies against phospho‐Chk1 (1/1,000, Ser345, Cell Signaling Technology, #2348), p53 (1/1,500, Cell Signaling Technology, #2524), and β‐actin (1/10,000, Sigma‐Aldrich, #A1978), and with polyclonal rabbit antibodies against phospho‐H2AX (1/1500, Ser139, Cell Signaling Technology, #2577), phospho‐p53 (1/1500, Ser15, Cell Signaling Technology, #9289), and cleaved caspase‐3 (1/1,000, Asp175, Cell Signaling Technology, #9661), prior to incubation with horseradish peroxidase‐conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, #115‐035‐144 and 111‐035‐144).

    Techniques: Inhibition, Immunolabeling

    Activation of the ATM‐Chk2‐p53 pathway upon CDDP treatment Confocal images showing the basal region of organ of Corti cultures treated with either culture medium alone or medium containing 10 μM CDDP for 1 day and immunolabeled for myosin 7A (red) and p‐ATM (green). Scale bar = 15 μm. Higher magnification images showing representative OHC and IHC nuclei from control and CDDP‐exposed cells. Scale bar = 5 μm. Histograms displaying green fluorescent signal intensity of x ‐projections of OHC and IHC nuclei from control and CDDP‐exposed cells. Quantification analysis of p‐ATM foci numbers in both OHCs and IHCs exposed to either culture medium alone (light blue and red lines for OHCs and IHCs, respectively) or 10 μM CDDP (blue and red lines for OHCs and IHCs, respectively; n = 50 nuclei per condition and per time point). Data are expressed as mean ± SEM. One‐way ANOVA test followed by post hoc Tukey's test (** P

    Journal: EMBO Molecular Medicine

    Article Title: Reversible p53 inhibition prevents cisplatin ototoxicity without blocking chemotherapeutic efficacy

    doi: 10.15252/emmm.201606230

    Figure Lengend Snippet: Activation of the ATM‐Chk2‐p53 pathway upon CDDP treatment Confocal images showing the basal region of organ of Corti cultures treated with either culture medium alone or medium containing 10 μM CDDP for 1 day and immunolabeled for myosin 7A (red) and p‐ATM (green). Scale bar = 15 μm. Higher magnification images showing representative OHC and IHC nuclei from control and CDDP‐exposed cells. Scale bar = 5 μm. Histograms displaying green fluorescent signal intensity of x ‐projections of OHC and IHC nuclei from control and CDDP‐exposed cells. Quantification analysis of p‐ATM foci numbers in both OHCs and IHCs exposed to either culture medium alone (light blue and red lines for OHCs and IHCs, respectively) or 10 μM CDDP (blue and red lines for OHCs and IHCs, respectively; n = 50 nuclei per condition and per time point). Data are expressed as mean ± SEM. One‐way ANOVA test followed by post hoc Tukey's test (** P

    Article Snippet: Blots were incubated with mouse monoclonal antibodies against phospho‐Chk1 (1/1,000, Ser345, Cell Signaling Technology, #2348), p53 (1/1,500, Cell Signaling Technology, #2524), and β‐actin (1/10,000, Sigma‐Aldrich, #A1978), and with polyclonal rabbit antibodies against phospho‐H2AX (1/1500, Ser139, Cell Signaling Technology, #2577), phospho‐p53 (1/1500, Ser15, Cell Signaling Technology, #9289), and cleaved caspase‐3 (1/1,000, Asp175, Cell Signaling Technology, #9661), prior to incubation with horseradish peroxidase‐conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, #115‐035‐144 and 111‐035‐144).

    Techniques: Activation Assay, Immunolabeling, Immunohistochemistry

    Intratympanic injection, tumor histology, and p53 genotyping and activation in vivo Shown is the custom‐made microendoscope. It consists of a fiber optic lens (10,000‐pixel resolution) for viewing and one catheter channel equipped with a fine needle for drug delivery. The blue arrowhead indicates the needle. Endoscopic view of the tympanic membrane (left image) and intratympanic injection through a fine needle (right image). The blue arrowhead indicates the needle. Representative scanned images of hematoxylin–eosin–safranin‐stained TP53 ‐mutant (HBCx‐14) tumor sections. Tumors were collected at day 21. Right panels: Higher magnification images show tumor features from the black boxed area in the left panels. The black arrowhead in the lower right panel indicates a remaining tumor cell. fs: fibrous scar, tc: tumor cells. Scale bars = left panels, 200 μm; right panels, 50 μm. Ratio of fibrous scar area/tumor cell area (sum of fibrous scar area/sum of tumor cell area). Note that combined treatments were much more efficient in replacing tumor cells by fibrous scar. The tumors were collected at day 21 ( n = 4 sections/tumor and 3–4 tumors/group). Data are expressed as mean ± SEM. One‐way ANOVA test followed by post hoc Tukey's test (** P = 0.002, *** P = 0.00008; CDDP versus DMSO or CDDP versus CDDP + PFT‐α). Representative PCR analysis of p53 expression from p53 wild‐type (wt), heterozygous (+/−), and knockout (−/−) mice. The 706‐bp band (neomycin cassette) is detected in p53 +/− and p53 −/− , although not in p53wt mice. By contrast, the 470‐bp band (p53 wild‐type gene) was only detected in p53wt and p53 +/− mice. Representative Western blots using antibodies against p‐p53 (serine 15) and actin in whole cochlear extracts from p53wt mice treated with CDDP for 0, 2, and 5 days. Note the increase in p53 phosphorylation in 2‐ and 5‐day CDDP‐treated cochleae.

    Journal: EMBO Molecular Medicine

    Article Title: Reversible p53 inhibition prevents cisplatin ototoxicity without blocking chemotherapeutic efficacy

    doi: 10.15252/emmm.201606230

    Figure Lengend Snippet: Intratympanic injection, tumor histology, and p53 genotyping and activation in vivo Shown is the custom‐made microendoscope. It consists of a fiber optic lens (10,000‐pixel resolution) for viewing and one catheter channel equipped with a fine needle for drug delivery. The blue arrowhead indicates the needle. Endoscopic view of the tympanic membrane (left image) and intratympanic injection through a fine needle (right image). The blue arrowhead indicates the needle. Representative scanned images of hematoxylin–eosin–safranin‐stained TP53 ‐mutant (HBCx‐14) tumor sections. Tumors were collected at day 21. Right panels: Higher magnification images show tumor features from the black boxed area in the left panels. The black arrowhead in the lower right panel indicates a remaining tumor cell. fs: fibrous scar, tc: tumor cells. Scale bars = left panels, 200 μm; right panels, 50 μm. Ratio of fibrous scar area/tumor cell area (sum of fibrous scar area/sum of tumor cell area). Note that combined treatments were much more efficient in replacing tumor cells by fibrous scar. The tumors were collected at day 21 ( n = 4 sections/tumor and 3–4 tumors/group). Data are expressed as mean ± SEM. One‐way ANOVA test followed by post hoc Tukey's test (** P = 0.002, *** P = 0.00008; CDDP versus DMSO or CDDP versus CDDP + PFT‐α). Representative PCR analysis of p53 expression from p53 wild‐type (wt), heterozygous (+/−), and knockout (−/−) mice. The 706‐bp band (neomycin cassette) is detected in p53 +/− and p53 −/− , although not in p53wt mice. By contrast, the 470‐bp band (p53 wild‐type gene) was only detected in p53wt and p53 +/− mice. Representative Western blots using antibodies against p‐p53 (serine 15) and actin in whole cochlear extracts from p53wt mice treated with CDDP for 0, 2, and 5 days. Note the increase in p53 phosphorylation in 2‐ and 5‐day CDDP‐treated cochleae.

    Article Snippet: Blots were incubated with mouse monoclonal antibodies against phospho‐Chk1 (1/1,000, Ser345, Cell Signaling Technology, #2348), p53 (1/1,500, Cell Signaling Technology, #2524), and β‐actin (1/10,000, Sigma‐Aldrich, #A1978), and with polyclonal rabbit antibodies against phospho‐H2AX (1/1500, Ser139, Cell Signaling Technology, #2577), phospho‐p53 (1/1500, Ser15, Cell Signaling Technology, #9289), and cleaved caspase‐3 (1/1,000, Asp175, Cell Signaling Technology, #9661), prior to incubation with horseradish peroxidase‐conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, #115‐035‐144 and 111‐035‐144).

    Techniques: Injection, Activation Assay, In Vivo, Staining, Mutagenesis, Polymerase Chain Reaction, Expressing, Knock-Out, Mouse Assay, Western Blot

    Effects of CDDP treatment on the DNA damage pathways, and ATM and p53 inhibition Representative PCR analysis of mATR and mChk1 expression in cochleae, testis, and kidney tissues from P3 mice. Water was used as a negative control. Representative Western blot analysis showing the level of Chk1 phosphorylation in protein extracts from cultured whole cochleae treated or not with 10 μM CDDP and from control and UV light‐exposed mouse embryonic fibroblasts (MEF). 3D images showing IHC and OHC nuclei from the basal region of the organ of Corti cultures treated with medium alone (control) or 10 μM CDDP for 1 day and immunolabeled for p‐ATM (green in C) or p‐Chk2 (green in D). Scale bar = 5 μm. 3D images showing OHC nuclei from the basal region of the organ of Corti cultures treated with medium alone, 10 μM KU55933, 10 μM CDDP, or 10 μM CDDP in combination with 10 μM KU55933 for 1 day and immunolabeled for p‐ATM (green). Scale bar = 5 μm. Quantification of p‐ATM foci number per nucleus in both inner (red bars) and outer (blue bars) hair cells for all treatment conditions ( n = 50 nuclei per condition and per time point). Data are expressed as mean ± SEM. One‐way ANOVA test followed by post hoc Tukey's test (** P ≤ 0.008, *** P ≤ 0.0006; CDDP versus control or CDDP versus CDDP + KU55933). Confocal images showing the basal region of organ of Corti cultures treated with medium alone, 100 μM PFT‐α, 10 μM CDDP, or 10 μM CDDP in combination with 100 μM PFT‐α for 5 days and immunolabeled for myosin 7A (red). Scale bar = 24 μm. Histograms representing the levels of surviving IHCs (red bars) and OHCs (blue bars) for all conditions after 5 days ( n = 5 cochleae per condition and per time point). Data are expressed as mean ± SEM. One‐way ANOVA test followed by post hoc Tukey's test (* P = 0.02, ** P ≤ 0.006, *** P = 0.0004; CDDP versus control, or CDDP versus CDDP + PFT‐α). Data information: All experiments were performed in triplicate.

    Journal: EMBO Molecular Medicine

    Article Title: Reversible p53 inhibition prevents cisplatin ototoxicity without blocking chemotherapeutic efficacy

    doi: 10.15252/emmm.201606230

    Figure Lengend Snippet: Effects of CDDP treatment on the DNA damage pathways, and ATM and p53 inhibition Representative PCR analysis of mATR and mChk1 expression in cochleae, testis, and kidney tissues from P3 mice. Water was used as a negative control. Representative Western blot analysis showing the level of Chk1 phosphorylation in protein extracts from cultured whole cochleae treated or not with 10 μM CDDP and from control and UV light‐exposed mouse embryonic fibroblasts (MEF). 3D images showing IHC and OHC nuclei from the basal region of the organ of Corti cultures treated with medium alone (control) or 10 μM CDDP for 1 day and immunolabeled for p‐ATM (green in C) or p‐Chk2 (green in D). Scale bar = 5 μm. 3D images showing OHC nuclei from the basal region of the organ of Corti cultures treated with medium alone, 10 μM KU55933, 10 μM CDDP, or 10 μM CDDP in combination with 10 μM KU55933 for 1 day and immunolabeled for p‐ATM (green). Scale bar = 5 μm. Quantification of p‐ATM foci number per nucleus in both inner (red bars) and outer (blue bars) hair cells for all treatment conditions ( n = 50 nuclei per condition and per time point). Data are expressed as mean ± SEM. One‐way ANOVA test followed by post hoc Tukey's test (** P ≤ 0.008, *** P ≤ 0.0006; CDDP versus control or CDDP versus CDDP + KU55933). Confocal images showing the basal region of organ of Corti cultures treated with medium alone, 100 μM PFT‐α, 10 μM CDDP, or 10 μM CDDP in combination with 100 μM PFT‐α for 5 days and immunolabeled for myosin 7A (red). Scale bar = 24 μm. Histograms representing the levels of surviving IHCs (red bars) and OHCs (blue bars) for all conditions after 5 days ( n = 5 cochleae per condition and per time point). Data are expressed as mean ± SEM. One‐way ANOVA test followed by post hoc Tukey's test (* P = 0.02, ** P ≤ 0.006, *** P = 0.0004; CDDP versus control, or CDDP versus CDDP + PFT‐α). Data information: All experiments were performed in triplicate.

    Article Snippet: Blots were incubated with mouse monoclonal antibodies against phospho‐Chk1 (1/1,000, Ser345, Cell Signaling Technology, #2348), p53 (1/1,500, Cell Signaling Technology, #2524), and β‐actin (1/10,000, Sigma‐Aldrich, #A1978), and with polyclonal rabbit antibodies against phospho‐H2AX (1/1500, Ser139, Cell Signaling Technology, #2577), phospho‐p53 (1/1500, Ser15, Cell Signaling Technology, #9289), and cleaved caspase‐3 (1/1,000, Asp175, Cell Signaling Technology, #9661), prior to incubation with horseradish peroxidase‐conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, #115‐035‐144 and 111‐035‐144).

    Techniques: Inhibition, Polymerase Chain Reaction, Expressing, Mouse Assay, Negative Control, Western Blot, Cell Culture, Immunohistochemistry, Immunolabeling

    Genetic and pharmacological deletion of p53 prevents loss of hearing and hair cells in adult mice Representative auditory brainstem response (ABR) waveforms evoked by 16 kHz tone bursts in p53wt mice treated with saline (black plot) or CDDP (green plot), and p53 −/− mice treated with saline (light blue plot) or CDDP (dark blue plot) for 5 days. ABR thresholds recorded in p53wt mice before (gray plot) and after 5 days of saline (black plot) or CDDP treatments (green plot), and ABR thresholds recorded in p53 −/− mice before (light gray plot), and after 5 days of saline (light blue plot) or CDDP treatment (dark blue plot). Saline‐treated group: n = 7; CDDP‐treated group: n = 12. Mean ABR threshold from 4 kHz to 32 kHz derived from (B). One‐way ANOVA test followed by post hoc Tukey's test (** P ≤ 0.008; p53wt + CDDP, d5 versus p53wt, before or p53wt + CDDP, d5 versus p53 −/− + CDDP, d5). Representative scanning electron microscopy micrographs showing the basal regions of cochleae from CDDP‐treated p53wt and p53 −/− mice after 5 days. Scale bar = 15 μm. Cytocochleograms representing the percentage of surviving hair cells in four cochlear regions located at 1.1, 2.6, 3.5, or 4.1 mm from the cochlear apex provided from saline‐treated p53wt mice (black bars), CDDP‐treated p53wt mice (green bars), or p53 −/− mice (blue bars), after 5 days ( n = 5 per group). Kruskal–Wallis test followed by post hoc Dunn's test (** P ≤ 0.008, p53wt + CDDP, d5 versus p53wt + saline, d5 or p53wt + CDDP, d5 versus p53 −/− + CDDP, d5). ABR thresholds from p53wt mice recorded prior to (gray plot) or after 5 days systemic treatment with: DMSO (yellow plot), CDDP + DMSO (green plot), CDDP + PFT‐α (pink plot). DMSO‐treated group: n = 7; CDDP + DMSO‐treated group: n = 12; CDDP + PFT‐α‐treated group: n = 12. Mean ABR threshold from 4 to 32 kHz derived from (F). One‐way ANOVA test followed by post hoc Tukey's test (*** P ≤ 0.0005; CDDP + DMSO, d5 versus before or CDDP + DMSO, d5 versus CDDP + PFT‐α, d5). Cytocochleograms representing the percentage of surviving hair cells in four cochlear regions located at 1.1, 2.6, 3.5, or 4.1 mm from the cochlear apical end taken from p53wt mice treated with: DMSO (yellow bars), CDDP + DMSO (green bars), CDDP + PFT‐α (pink bars), after 5 days ( n = 5 per group). Kruskal–Wallis test followed by post hoc Dunn's test (** P ≤ 0.008; CDDP + DMSO, d5 versus DMSO, d5 or CDDP + DMSO, d5 versus CDDP + PFT‐α, d5). ABR thresholds from p53wt mice recorded prior to (gray plot and black plot for left and right ear, respectively) or after 5 days of systemic treatment with CDDP plus intratympanic injection of DMSO into the left ear (green plot) and of PFT‐α into the right ear (pink plot). n = 14 per group. Mean ABR threshold from 4 to 32 kHz derived from (I) ( n = 14). One‐way ANOVA test followed by post hoc Tukey's test (*** P = 0.0003; CDDP + left ear, DMSO versus before, left ear or CDDP + left ear, DMSO versus CDDP + right ear, PFT‐α). Cytocochleograms representing the percentage of surviving hair cells in four cochlear regions located at 1.1, 2.6, 3.5, or 4.1 mm from the cochlear apical end taken from the DMSO‐treated left ear (green bars) and PFT‐α‐treated right ear (pink bars) of the same CDDP‐treated p53wt mice after 5 days ( n = 5 per group). Kruskal–Wallis test followed by post hoc Dunn's test (** P ≤ 0.008; CDDP + right ear, PFT‐α versus CDDP + left ear, DMSO). Data information: Data are expressed as mean ± SEM.

    Journal: EMBO Molecular Medicine

    Article Title: Reversible p53 inhibition prevents cisplatin ototoxicity without blocking chemotherapeutic efficacy

    doi: 10.15252/emmm.201606230

    Figure Lengend Snippet: Genetic and pharmacological deletion of p53 prevents loss of hearing and hair cells in adult mice Representative auditory brainstem response (ABR) waveforms evoked by 16 kHz tone bursts in p53wt mice treated with saline (black plot) or CDDP (green plot), and p53 −/− mice treated with saline (light blue plot) or CDDP (dark blue plot) for 5 days. ABR thresholds recorded in p53wt mice before (gray plot) and after 5 days of saline (black plot) or CDDP treatments (green plot), and ABR thresholds recorded in p53 −/− mice before (light gray plot), and after 5 days of saline (light blue plot) or CDDP treatment (dark blue plot). Saline‐treated group: n = 7; CDDP‐treated group: n = 12. Mean ABR threshold from 4 kHz to 32 kHz derived from (B). One‐way ANOVA test followed by post hoc Tukey's test (** P ≤ 0.008; p53wt + CDDP, d5 versus p53wt, before or p53wt + CDDP, d5 versus p53 −/− + CDDP, d5). Representative scanning electron microscopy micrographs showing the basal regions of cochleae from CDDP‐treated p53wt and p53 −/− mice after 5 days. Scale bar = 15 μm. Cytocochleograms representing the percentage of surviving hair cells in four cochlear regions located at 1.1, 2.6, 3.5, or 4.1 mm from the cochlear apex provided from saline‐treated p53wt mice (black bars), CDDP‐treated p53wt mice (green bars), or p53 −/− mice (blue bars), after 5 days ( n = 5 per group). Kruskal–Wallis test followed by post hoc Dunn's test (** P ≤ 0.008, p53wt + CDDP, d5 versus p53wt + saline, d5 or p53wt + CDDP, d5 versus p53 −/− + CDDP, d5). ABR thresholds from p53wt mice recorded prior to (gray plot) or after 5 days systemic treatment with: DMSO (yellow plot), CDDP + DMSO (green plot), CDDP + PFT‐α (pink plot). DMSO‐treated group: n = 7; CDDP + DMSO‐treated group: n = 12; CDDP + PFT‐α‐treated group: n = 12. Mean ABR threshold from 4 to 32 kHz derived from (F). One‐way ANOVA test followed by post hoc Tukey's test (*** P ≤ 0.0005; CDDP + DMSO, d5 versus before or CDDP + DMSO, d5 versus CDDP + PFT‐α, d5). Cytocochleograms representing the percentage of surviving hair cells in four cochlear regions located at 1.1, 2.6, 3.5, or 4.1 mm from the cochlear apical end taken from p53wt mice treated with: DMSO (yellow bars), CDDP + DMSO (green bars), CDDP + PFT‐α (pink bars), after 5 days ( n = 5 per group). Kruskal–Wallis test followed by post hoc Dunn's test (** P ≤ 0.008; CDDP + DMSO, d5 versus DMSO, d5 or CDDP + DMSO, d5 versus CDDP + PFT‐α, d5). ABR thresholds from p53wt mice recorded prior to (gray plot and black plot for left and right ear, respectively) or after 5 days of systemic treatment with CDDP plus intratympanic injection of DMSO into the left ear (green plot) and of PFT‐α into the right ear (pink plot). n = 14 per group. Mean ABR threshold from 4 to 32 kHz derived from (I) ( n = 14). One‐way ANOVA test followed by post hoc Tukey's test (*** P = 0.0003; CDDP + left ear, DMSO versus before, left ear or CDDP + left ear, DMSO versus CDDP + right ear, PFT‐α). Cytocochleograms representing the percentage of surviving hair cells in four cochlear regions located at 1.1, 2.6, 3.5, or 4.1 mm from the cochlear apical end taken from the DMSO‐treated left ear (green bars) and PFT‐α‐treated right ear (pink bars) of the same CDDP‐treated p53wt mice after 5 days ( n = 5 per group). Kruskal–Wallis test followed by post hoc Dunn's test (** P ≤ 0.008; CDDP + right ear, PFT‐α versus CDDP + left ear, DMSO). Data information: Data are expressed as mean ± SEM.

    Article Snippet: Blots were incubated with mouse monoclonal antibodies against phospho‐Chk1 (1/1,000, Ser345, Cell Signaling Technology, #2348), p53 (1/1,500, Cell Signaling Technology, #2524), and β‐actin (1/10,000, Sigma‐Aldrich, #A1978), and with polyclonal rabbit antibodies against phospho‐H2AX (1/1500, Ser139, Cell Signaling Technology, #2577), phospho‐p53 (1/1500, Ser15, Cell Signaling Technology, #9289), and cleaved caspase‐3 (1/1,000, Asp175, Cell Signaling Technology, #9661), prior to incubation with horseradish peroxidase‐conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, #115‐035‐144 and 111‐035‐144).

    Techniques: Mouse Assay, Derivative Assay, Electron Microscopy, Injection

    PFT‐α enhances CDDP‐induced anti‐angiogenesis and suppresses autophagy selectively in TP53‐ mutant tumors Representative confocal images of microvessels in transversal tumor sections from HBCx‐14 ( TP53 ‐mutant) tumors treated with either DMSO, CDDP, or a combination of CDDP and PFT‐α. The sections were immunolabeled for CD31 (red) and viewed with a 20× objective. The basal‐like breast cancer cells were immunolabeled in green with an antibody against cytokeratin 5 and 8. Upper right panel is a 2D projection from the white boxed area in upper left panel. The red area corresponds to CD31‐labeled endothelial area, and the white area represents the lumen area. The white line shows the vessel perimeter. Scale bars = left panels, 50 μm; right panels, 35 μm. The tumor samples were collected at day 21. Histograms representing the percentage of vascular area calculated using 2D reconstruction image analysis and the formula: vessel/vascular area = area of CD31‐positive objects + lumen area per field area × 100%. Both HBCx‐14 ( TP53 ‐mutant) and HBCx‐90 ( TP53 wt) tumors from the different treated groups were collected at day 21 ( n = 4 sections/tumor and 3–4 tumors/group). Data are expressed as mean ± SEM. Kruskal–Wallis test followed by post hoc Dunn's test (*** P ≤ 0.0004; CDDP versus DMSO or CDDP versus CDDP + PFT‐α). Representative confocal images of cleaved caspase‐3‐positive cells (red) in the transversal tumor sections and viewed with a 20× objective. The stromal compartments were immunolabeled in green with an antibody against vimentin. HBCx‐14 ( TP53 ‐mutant) tumors from the different treated groups were collected at day 18. Scale bar = 50 μm. c‐cas‐3: cleaved caspase‐3. Representative Western blot analysis using antibodies against β‐actin, p53, and p21 in tumor extracts from HBCx‐14 ( TP53 ‐mutant) and HBCx‐90 ( TP53 wt) tumors treated with either DMSO or CDDP and collected at day 18. Note the higher CDDP‐induced increase in p53 and p21 expression in TP53 wt HBCx‐90 tumors. Representative Western blot analysis using antibodies against β‐actin, p‐Chk1, Beclin 1, LC3‐I/II, and Rab7 in tumor extracts from HBCx‐14 ( TP53 ‐mutant) and HBCx‐90 ( TP53 wt) tumors treated with either DMSO, PFT‐α, CDDP, or a combination of CDDP and PFT‐α. The tumor samples were collected at day 18. Histograms representing the levels of Beclin 1, LC3‐II, and Rab7 in HBCx‐14 and HBCx‐90 tumors treated with the different regimens ( n = 3–4 tumors per group, all experiments were performed in triplicate). Actin served as a loading control. Data are expressed as mean ± SEM. One‐way ANOVA test followed by post hoc Tukey's test (G: * P = 0.03, ** P = 0.006, *** P ≤ 0.0004; H: * P = 0.02, *** P = 0.0005; I: * P = 0.03, *** P ≤ 0.0006; CDDP versus DMSO or CDDP versus CDDP + PFT‐α).

    Journal: EMBO Molecular Medicine

    Article Title: Reversible p53 inhibition prevents cisplatin ototoxicity without blocking chemotherapeutic efficacy

    doi: 10.15252/emmm.201606230

    Figure Lengend Snippet: PFT‐α enhances CDDP‐induced anti‐angiogenesis and suppresses autophagy selectively in TP53‐ mutant tumors Representative confocal images of microvessels in transversal tumor sections from HBCx‐14 ( TP53 ‐mutant) tumors treated with either DMSO, CDDP, or a combination of CDDP and PFT‐α. The sections were immunolabeled for CD31 (red) and viewed with a 20× objective. The basal‐like breast cancer cells were immunolabeled in green with an antibody against cytokeratin 5 and 8. Upper right panel is a 2D projection from the white boxed area in upper left panel. The red area corresponds to CD31‐labeled endothelial area, and the white area represents the lumen area. The white line shows the vessel perimeter. Scale bars = left panels, 50 μm; right panels, 35 μm. The tumor samples were collected at day 21. Histograms representing the percentage of vascular area calculated using 2D reconstruction image analysis and the formula: vessel/vascular area = area of CD31‐positive objects + lumen area per field area × 100%. Both HBCx‐14 ( TP53 ‐mutant) and HBCx‐90 ( TP53 wt) tumors from the different treated groups were collected at day 21 ( n = 4 sections/tumor and 3–4 tumors/group). Data are expressed as mean ± SEM. Kruskal–Wallis test followed by post hoc Dunn's test (*** P ≤ 0.0004; CDDP versus DMSO or CDDP versus CDDP + PFT‐α). Representative confocal images of cleaved caspase‐3‐positive cells (red) in the transversal tumor sections and viewed with a 20× objective. The stromal compartments were immunolabeled in green with an antibody against vimentin. HBCx‐14 ( TP53 ‐mutant) tumors from the different treated groups were collected at day 18. Scale bar = 50 μm. c‐cas‐3: cleaved caspase‐3. Representative Western blot analysis using antibodies against β‐actin, p53, and p21 in tumor extracts from HBCx‐14 ( TP53 ‐mutant) and HBCx‐90 ( TP53 wt) tumors treated with either DMSO or CDDP and collected at day 18. Note the higher CDDP‐induced increase in p53 and p21 expression in TP53 wt HBCx‐90 tumors. Representative Western blot analysis using antibodies against β‐actin, p‐Chk1, Beclin 1, LC3‐I/II, and Rab7 in tumor extracts from HBCx‐14 ( TP53 ‐mutant) and HBCx‐90 ( TP53 wt) tumors treated with either DMSO, PFT‐α, CDDP, or a combination of CDDP and PFT‐α. The tumor samples were collected at day 18. Histograms representing the levels of Beclin 1, LC3‐II, and Rab7 in HBCx‐14 and HBCx‐90 tumors treated with the different regimens ( n = 3–4 tumors per group, all experiments were performed in triplicate). Actin served as a loading control. Data are expressed as mean ± SEM. One‐way ANOVA test followed by post hoc Tukey's test (G: * P = 0.03, ** P = 0.006, *** P ≤ 0.0004; H: * P = 0.02, *** P = 0.0005; I: * P = 0.03, *** P ≤ 0.0006; CDDP versus DMSO or CDDP versus CDDP + PFT‐α).

    Article Snippet: Blots were incubated with mouse monoclonal antibodies against phospho‐Chk1 (1/1,000, Ser345, Cell Signaling Technology, #2348), p53 (1/1,500, Cell Signaling Technology, #2524), and β‐actin (1/10,000, Sigma‐Aldrich, #A1978), and with polyclonal rabbit antibodies against phospho‐H2AX (1/1500, Ser139, Cell Signaling Technology, #2577), phospho‐p53 (1/1500, Ser15, Cell Signaling Technology, #9289), and cleaved caspase‐3 (1/1,000, Asp175, Cell Signaling Technology, #9661), prior to incubation with horseradish peroxidase‐conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, #115‐035‐144 and 111‐035‐144).

    Techniques: Mutagenesis, Immunolabeling, Labeling, Western Blot, Expressing

    PFT‐α protects cochlea from ototoxicity without compromising and even enhancing CDDP anti‐tumor efficacy in patient‐derived breast cancer xenograft mice ABR thresholds recorded prior to (gray plot) or at day 28 in HBCx‐90 ( TP53 wt)‐bearing mice received systemic treatment with: DMSO (black plot), PFT‐α (yellow plot), CDDP (green plot), CDDP + PFT‐α (pink plot). Before: n = 20; DMSO: n = 5; PFT‐α: n = 5; CDDP: n = 5; CDDP + PFT‐α: n = 5. Mean ABR threshold from 4 to 32 kHz derived from (A). One‐way ANOVA test followed by post hoc Tukey's test (*** P = 0.0002; CDDP versus before, CDDP versus CDDP + PFT‐α). Cytocochleograms representing the percentage of surviving hair cells in three cochlear regions located at 1.1, 2.6, and 3.5 mm from the cochlear apical end. These cochleae ( n = 5 per group) were collected at 28 days from the beginning of treatment with DMSO (black bars), PFT‐α (yellow bars), CDDP (green bars), or CDDP + PFT‐α (pink bars). Kruskal–Wallis test followed by post hoc Dunn's test (** P ≤ 0.007; CDDP, d28 versus DMSO, d28 or CDDP, d28 versus CDDP + PFT‐α, d28). Tumor growth curves alongside images of dissected tumors collected on day 28. Note the partial inhibition of growth in CDDP and CDDP + PFT‐α‐treated mice. 0, 5, 7 and 14 days: DMSO‐ or PFT‐α‐treated group: n = 10; CDDP‐ or CDDP + PFT‐α‐treated group: n = 11. 21 days: DMSO‐ or PFT‐α‐treated group: n = 7; CDDP‐ or CDDP + PFT‐α‐treated group: n = 8. 28 days: n = 5 per group. ABR thresholds recorded prior to (gray plot) or at day 35 in HBCx‐14 ( TP53 ‐mutant)‐bearing mice received systemic treatment with DMSO (black plot), PFT‐α (yellow plot), CDDP (green plot), CDDP + PFT‐α (pink plot). Before: n = 30; DMSO: n = 5; PFT‐α: n = 5; CDDP: n = 10; CDDP + PFT‐α: n = 10. Mean ABR threshold from 4 kHz to 32 kHz derived from (E). One‐way ANOVA test followed by post hoc Tukey's test (*** P = 0.0003; CDDP versus before or CDDP versus CDDP + PFT‐α). Cytocochleograms representing the percentage of surviving hair cells in three cochlear regions located at 1.1, 2.6, and 3.5 mm from the cochlear apical end. These cochleae ( n = 5 per group) were collected at 35 days from the beginning of treatment with DMSO (black bars), PFT‐α (yellow bars), CDDP (green bars), or CDDP + PFT‐α (pink bars). Kruskal–Wallis test followed by post hoc Dunn's test (** P ≤ 0.008; CDDP, d35 versus DMSO, d35 or CDDP, d35 versus CDDP + PFT‐α, d35). Tumor growth curves alongside images of dissected tumors collected on day 35. Note the complete disappearance of tumors at day 35 and the significantly reduced tumor regrowth up to day 70 in CDDP + PFT‐α‐treated mice. 0, 7 and 14 days: DMSO‐ or PFT‐α‐treated group: n = 11; CDDP‐treated group: n = 16; CDDP + PFT‐α‐treated group: n = 17. 21 days: DMSO‐ or PFT‐α‐treated group: n = 8; CDDP‐treated group: n = 13; CDDP + PFT‐α‐treated group: n = 14. 35 days: DMSO‐ or PFT‐α‐treated group: n = 5; CDDP‐treated group: n = 10; CDDP + PFT‐α‐treated group: n = 10. 42, 56 and 70 days: CDDP‐ or CDDP + PFT‐α‐treated group n = 5 per group. Data information: Data are expressed as mean ± SEM.

    Journal: EMBO Molecular Medicine

    Article Title: Reversible p53 inhibition prevents cisplatin ototoxicity without blocking chemotherapeutic efficacy

    doi: 10.15252/emmm.201606230

    Figure Lengend Snippet: PFT‐α protects cochlea from ototoxicity without compromising and even enhancing CDDP anti‐tumor efficacy in patient‐derived breast cancer xenograft mice ABR thresholds recorded prior to (gray plot) or at day 28 in HBCx‐90 ( TP53 wt)‐bearing mice received systemic treatment with: DMSO (black plot), PFT‐α (yellow plot), CDDP (green plot), CDDP + PFT‐α (pink plot). Before: n = 20; DMSO: n = 5; PFT‐α: n = 5; CDDP: n = 5; CDDP + PFT‐α: n = 5. Mean ABR threshold from 4 to 32 kHz derived from (A). One‐way ANOVA test followed by post hoc Tukey's test (*** P = 0.0002; CDDP versus before, CDDP versus CDDP + PFT‐α). Cytocochleograms representing the percentage of surviving hair cells in three cochlear regions located at 1.1, 2.6, and 3.5 mm from the cochlear apical end. These cochleae ( n = 5 per group) were collected at 28 days from the beginning of treatment with DMSO (black bars), PFT‐α (yellow bars), CDDP (green bars), or CDDP + PFT‐α (pink bars). Kruskal–Wallis test followed by post hoc Dunn's test (** P ≤ 0.007; CDDP, d28 versus DMSO, d28 or CDDP, d28 versus CDDP + PFT‐α, d28). Tumor growth curves alongside images of dissected tumors collected on day 28. Note the partial inhibition of growth in CDDP and CDDP + PFT‐α‐treated mice. 0, 5, 7 and 14 days: DMSO‐ or PFT‐α‐treated group: n = 10; CDDP‐ or CDDP + PFT‐α‐treated group: n = 11. 21 days: DMSO‐ or PFT‐α‐treated group: n = 7; CDDP‐ or CDDP + PFT‐α‐treated group: n = 8. 28 days: n = 5 per group. ABR thresholds recorded prior to (gray plot) or at day 35 in HBCx‐14 ( TP53 ‐mutant)‐bearing mice received systemic treatment with DMSO (black plot), PFT‐α (yellow plot), CDDP (green plot), CDDP + PFT‐α (pink plot). Before: n = 30; DMSO: n = 5; PFT‐α: n = 5; CDDP: n = 10; CDDP + PFT‐α: n = 10. Mean ABR threshold from 4 kHz to 32 kHz derived from (E). One‐way ANOVA test followed by post hoc Tukey's test (*** P = 0.0003; CDDP versus before or CDDP versus CDDP + PFT‐α). Cytocochleograms representing the percentage of surviving hair cells in three cochlear regions located at 1.1, 2.6, and 3.5 mm from the cochlear apical end. These cochleae ( n = 5 per group) were collected at 35 days from the beginning of treatment with DMSO (black bars), PFT‐α (yellow bars), CDDP (green bars), or CDDP + PFT‐α (pink bars). Kruskal–Wallis test followed by post hoc Dunn's test (** P ≤ 0.008; CDDP, d35 versus DMSO, d35 or CDDP, d35 versus CDDP + PFT‐α, d35). Tumor growth curves alongside images of dissected tumors collected on day 35. Note the complete disappearance of tumors at day 35 and the significantly reduced tumor regrowth up to day 70 in CDDP + PFT‐α‐treated mice. 0, 7 and 14 days: DMSO‐ or PFT‐α‐treated group: n = 11; CDDP‐treated group: n = 16; CDDP + PFT‐α‐treated group: n = 17. 21 days: DMSO‐ or PFT‐α‐treated group: n = 8; CDDP‐treated group: n = 13; CDDP + PFT‐α‐treated group: n = 14. 35 days: DMSO‐ or PFT‐α‐treated group: n = 5; CDDP‐treated group: n = 10; CDDP + PFT‐α‐treated group: n = 10. 42, 56 and 70 days: CDDP‐ or CDDP + PFT‐α‐treated group n = 5 per group. Data information: Data are expressed as mean ± SEM.

    Article Snippet: Blots were incubated with mouse monoclonal antibodies against phospho‐Chk1 (1/1,000, Ser345, Cell Signaling Technology, #2348), p53 (1/1,500, Cell Signaling Technology, #2524), and β‐actin (1/10,000, Sigma‐Aldrich, #A1978), and with polyclonal rabbit antibodies against phospho‐H2AX (1/1500, Ser139, Cell Signaling Technology, #2577), phospho‐p53 (1/1500, Ser15, Cell Signaling Technology, #9289), and cleaved caspase‐3 (1/1,000, Asp175, Cell Signaling Technology, #9661), prior to incubation with horseradish peroxidase‐conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, #115‐035‐144 and 111‐035‐144).

    Techniques: Derivative Assay, Mouse Assay, Inhibition, Mutagenesis

    Accumulation of proteasomal targets and polyubiquitinated proteins in Sesn2 KO MLFs. A , expression of c-myc and p53 in WT and Sesn2 KO MLFs after stimulation with PDGF-BB for the indicated time intervals. B , polyubiquitinated protein levels in WT and

    Journal: The Journal of Biological Chemistry

    Article Title: Sestrin 2 Protein Regulates Platelet-derived Growth Factor Receptor β (Pdgfrβ) Expression by Modulating Proteasomal and Nrf2 Transcription Factor Functions *

    doi: 10.1074/jbc.M114.632133

    Figure Lengend Snippet: Accumulation of proteasomal targets and polyubiquitinated proteins in Sesn2 KO MLFs. A , expression of c-myc and p53 in WT and Sesn2 KO MLFs after stimulation with PDGF-BB for the indicated time intervals. B , polyubiquitinated protein levels in WT and

    Article Snippet: Rabbit monoclonal antibodies against DJ1, GAPDH, α-tubulin, hemoxygenase 1 (Ho1; P109), Keap1 (D6B12), MAP1LC3B (D11), and Pdgfrβ (28E1; used for Western blot) and mouse monoclonal antibody against p53 (1C12) were from Cell Signaling (Danvers, MA).

    Techniques: Expressing

    EGCG inhibits cisplatin-mediated apoptosis and inflammatory response in rat cochlea. ( a ) Male Wistar rats were pretreated with oral EGCG (100 mg/kg body weight) for 24 h, followed by cisplatin (11 mg/kg) and daily oral EGCG treatments were continued for an additional 3 days and killed on day 4. Cochleas were collected, fixed, decalcified and processed for mid-modiolar sections. Sections were used for TUNEL staining (red) along with phalloidin immunostaining (green). Cisplatin increased TUNEL-positive nuclei (red) in the OC, SL, SV and SGN, which was blocked by pre-treatment with oral EGCG. Scale bar represents 100 μ m. ( b ) In the OC region, TUNEL-positive nuclei (red) was observed in the OHCs, DCs, IPCs and OPCs of cisplatin-treated cochlea. No distinct TUNEL-positive nuclei were observed in IHCs and IPhCs. Oral EGCG inhibited the TUNEL-positive response induced by cisplatin in the OC. Scale bar represents 25 μ m. ( c ) Cochleae were collected for real-time RT-PCR to determine the levels of COX2 , iNOS , TNF-α , NOX3 , p53 , Bax , Bcl-2 and STAT1 mRNAs. The figures in ( a ) and ( b ) are representatives of cochleas from different groups of four rats each. Data presented in ( c ) represent the mean±S.E.M. of cochleas from four animals. Asterisk (*) indicates statistically significant difference from vehicle, while (**) indicates significant difference from the cisplatin group ( P

    Journal: Cell Death & Disease

    Article Title: Epigallocatechin-3-gallate, a prototypic chemopreventative agent for protection against cisplatin-based ototoxicity

    doi: 10.1038/cddis.2017.314

    Figure Lengend Snippet: EGCG inhibits cisplatin-mediated apoptosis and inflammatory response in rat cochlea. ( a ) Male Wistar rats were pretreated with oral EGCG (100 mg/kg body weight) for 24 h, followed by cisplatin (11 mg/kg) and daily oral EGCG treatments were continued for an additional 3 days and killed on day 4. Cochleas were collected, fixed, decalcified and processed for mid-modiolar sections. Sections were used for TUNEL staining (red) along with phalloidin immunostaining (green). Cisplatin increased TUNEL-positive nuclei (red) in the OC, SL, SV and SGN, which was blocked by pre-treatment with oral EGCG. Scale bar represents 100 μ m. ( b ) In the OC region, TUNEL-positive nuclei (red) was observed in the OHCs, DCs, IPCs and OPCs of cisplatin-treated cochlea. No distinct TUNEL-positive nuclei were observed in IHCs and IPhCs. Oral EGCG inhibited the TUNEL-positive response induced by cisplatin in the OC. Scale bar represents 25 μ m. ( c ) Cochleae were collected for real-time RT-PCR to determine the levels of COX2 , iNOS , TNF-α , NOX3 , p53 , Bax , Bcl-2 and STAT1 mRNAs. The figures in ( a ) and ( b ) are representatives of cochleas from different groups of four rats each. Data presented in ( c ) represent the mean±S.E.M. of cochleas from four animals. Asterisk (*) indicates statistically significant difference from vehicle, while (**) indicates significant difference from the cisplatin group ( P

    Article Snippet: Antibodies Various primary antibodies (with catalog numbers and companies) used were as follows: p-ERK1/2 (no. 7976), ERK1/2 (no. 93), STAT1 (no. 592), Na+/K+ ATPase α 1 (no. 21712) STAT3 (no. 8019), Bcl-xL (no. 8392), β -actin (no. 69879) from Santa Cruz Biotechnology (Dallas, TX, USA); JAK2 (no. 3230), pJAK2 (no. 3776), p-STAT3 Tyr705 (no. 4113 S), p-STAT1 Ser727 (no. 9177 S), p53 (no. 2524), Bcl-2 (no. 2876), cleaved caspase-3 (no. 9661), Bax (no. 2772), TNFα (no. 3707 S), Cox2 (no. 4842) from Cell Signaling Technology (Danvers, MA, USA); myosin VIIa rabbit polyclonal (no. 25-6790) from Proteus Biosciences (Ramona, CA, USA); CtBP2 mouse IgG1 (no. 612044) from BD Biosciences (San Jose, CA, USA); Alexa Fluor 488 Phalloidin (no. A-12379) from Thermo Fisher Scientific (Waltham, MA, USA).

    Techniques: TUNEL Assay, Staining, Immunostaining, Quantitative RT-PCR

    Evidence for a role of p53 and mTOR signalling during physiological competition during embryogenesis. a , b Wild-type embryos were cultured in DMSO or 200 uM pan-caspase inhibitor for 16–18 h and cell death was assessed by TUNEL staining. c Wild-type embryos were cultured in DMSO or caspase inhibitors and number of cells in the medial plane of the epiblast was quantified; caspase inhibition results in a 35% increase in the average number of cells counted in the medial plane of the epiblast (DMSO n = 8, average 28.9 cells; caspase inhibitor n = 10, average 39.1 cells). d Wild-type embryos cultured overnight in DMSO or caspase inhibitors were stained for pS6 S240/244 . e In each embryo, the percentage of cells below a ‘low pS6 cutoff’ in the medial plane of the epiblast was quantified (as shown in Supplementary Fig. 8A ): percentage of low pS6 cells increases from 2.8% in DMSO treated embryos to 41.5% in caspase-inhibitor-treated embryos. Data are collated from three litters (DMSO n = 13; caspase inhibitor n = 15). f Magnification of DMSO- or caspase inhibitor-treated embryos stained for pS6 and total S6. The boundary between the visceral endoderm (VE) and epiblast (epi) is indicated with a dashed line. g In each embryo, the percentage of cells below a ‘low pS6:S6’ cutoff (as shown in Supplementary Fig. 8B ) was quantified: percentage of low pS6:S6 cells per embryo increases from 4% in DMSO-treated embryos to 38% in caspase-inhibitor-treated embryos. Data are collated from one litter (DMSO n = 5; caspase inhibitor n = 5). h p53 expression in 5.5 dpc embryos detected by immunofluorescence. i Embryos were treated with DMSO or Nutlin-3a for 6 h and levels of p53 and pS6 were analysed by immunofluorescence and the correlation between p53 and pS6 levels were quantified at the single-cell level ( j ). Scale bars are 40 μm ( a , i ), 25 μm ( b ) or 20 μm ( f , h ). Error bars denote SD ( b , c ) or SEM ( e , g ). ** p

    Journal: Nature Communications

    Article Title: P53 and mTOR signalling determine fitness selection through cell competition during early mouse embryonic development

    doi: 10.1038/s41467-018-04167-y

    Figure Lengend Snippet: Evidence for a role of p53 and mTOR signalling during physiological competition during embryogenesis. a , b Wild-type embryos were cultured in DMSO or 200 uM pan-caspase inhibitor for 16–18 h and cell death was assessed by TUNEL staining. c Wild-type embryos were cultured in DMSO or caspase inhibitors and number of cells in the medial plane of the epiblast was quantified; caspase inhibition results in a 35% increase in the average number of cells counted in the medial plane of the epiblast (DMSO n = 8, average 28.9 cells; caspase inhibitor n = 10, average 39.1 cells). d Wild-type embryos cultured overnight in DMSO or caspase inhibitors were stained for pS6 S240/244 . e In each embryo, the percentage of cells below a ‘low pS6 cutoff’ in the medial plane of the epiblast was quantified (as shown in Supplementary Fig. 8A ): percentage of low pS6 cells increases from 2.8% in DMSO treated embryos to 41.5% in caspase-inhibitor-treated embryos. Data are collated from three litters (DMSO n = 13; caspase inhibitor n = 15). f Magnification of DMSO- or caspase inhibitor-treated embryos stained for pS6 and total S6. The boundary between the visceral endoderm (VE) and epiblast (epi) is indicated with a dashed line. g In each embryo, the percentage of cells below a ‘low pS6:S6’ cutoff (as shown in Supplementary Fig. 8B ) was quantified: percentage of low pS6:S6 cells per embryo increases from 4% in DMSO-treated embryos to 38% in caspase-inhibitor-treated embryos. Data are collated from one litter (DMSO n = 5; caspase inhibitor n = 5). h p53 expression in 5.5 dpc embryos detected by immunofluorescence. i Embryos were treated with DMSO or Nutlin-3a for 6 h and levels of p53 and pS6 were analysed by immunofluorescence and the correlation between p53 and pS6 levels were quantified at the single-cell level ( j ). Scale bars are 40 μm ( a , i ), 25 μm ( b ) or 20 μm ( f , h ). Error bars denote SD ( b , c ) or SEM ( e , g ). ** p

    Article Snippet: Antibodies used are as follows: pS6S240/244 #5264, S6 #2317, p53 #2524, p4E-BP1T37/46 #2855, total 4E-BP1 #9644, cleaved caspase-3 #9664, cleaved PARP #9544, pAKTT473 #4060, TSC2 #4308, β-actin #4970; all from Cell Signaling Technology and used at 1:1000 dilutions.

    Techniques: Cell Culture, TUNEL Assay, Staining, Inhibition, Expressing, Immunofluorescence

    p53 is upstream of mTOR pathway repression during ESC differentiation. a Total p53 levels in wild-type and Bmpr1a −/− cells in separate and co-culture at day 3 in competition were assessed by immunofluorescence and nuclear p53 levels were quantified ( b ). Scale bar = 50 μm. c Total p53 levels in wild-type and 4n cells in separate and co-culture were assessed by immunofluorescence and quantified. d Wild-type cells were infected with shRNAs targeting p53 and levels of mTOR pathway activity were assessed by western blot. e Wild-type cells were treated with p53 activator Nutlin-3a for 8 h and levels of mTOR pathway activity were assessed by western blot and quantified ( f ). g Fold change, apoptosis ( h ), and pS6 levels ( i ) in Bmpr1a −/− and Bmpr1a −/− ;p53 −/− (two independent clones) cultured separately and with wild-type cells (co-culture) from d0–d4. Apoptosis levels of Bmpr1a −/− ; p53 −/− were compared to Bmpr1a −/− ;Cas9 as previously shown (Fig. 3a ) n = 3 for all studies; error bars denote SEM. * p

    Journal: Nature Communications

    Article Title: P53 and mTOR signalling determine fitness selection through cell competition during early mouse embryonic development

    doi: 10.1038/s41467-018-04167-y

    Figure Lengend Snippet: p53 is upstream of mTOR pathway repression during ESC differentiation. a Total p53 levels in wild-type and Bmpr1a −/− cells in separate and co-culture at day 3 in competition were assessed by immunofluorescence and nuclear p53 levels were quantified ( b ). Scale bar = 50 μm. c Total p53 levels in wild-type and 4n cells in separate and co-culture were assessed by immunofluorescence and quantified. d Wild-type cells were infected with shRNAs targeting p53 and levels of mTOR pathway activity were assessed by western blot. e Wild-type cells were treated with p53 activator Nutlin-3a for 8 h and levels of mTOR pathway activity were assessed by western blot and quantified ( f ). g Fold change, apoptosis ( h ), and pS6 levels ( i ) in Bmpr1a −/− and Bmpr1a −/− ;p53 −/− (two independent clones) cultured separately and with wild-type cells (co-culture) from d0–d4. Apoptosis levels of Bmpr1a −/− ; p53 −/− were compared to Bmpr1a −/− ;Cas9 as previously shown (Fig. 3a ) n = 3 for all studies; error bars denote SEM. * p

    Article Snippet: Antibodies used are as follows: pS6S240/244 #5264, S6 #2317, p53 #2524, p4E-BP1T37/46 #2855, total 4E-BP1 #9644, cleaved caspase-3 #9664, cleaved PARP #9544, pAKTT473 #4060, TSC2 #4308, β-actin #4970; all from Cell Signaling Technology and used at 1:1000 dilutions.

    Techniques: Co-Culture Assay, Immunofluorescence, Infection, Activity Assay, Western Blot, Cell Culture