Journal: Otology & Neurotology

Article Title: Regulation of the p53/SLC7A11/GPX4 Pathway by Gentamicin Induces Ferroptosis in HEI-OC1 Cells

doi: 10.1097/MAO.0000000000004271

Figure Lengend Snippet: Gentamicin induces cellular ferroptosis through a p53-dependent mechanism. HEI-OC1 cells were treated with 10 μM PFT-α for 24 hours and then with 20 mM gentamicin for 24 hours. Protein expression levels of ( A ) p53 and ( B ) GPX4, and SLC7A11 were evaluated by Western blot. C , Intracellular ROS were detected by DCFH-DA fluorescent probe. D–F, Levels of Fe, MDA, and GSH were determined. * p < 0.05, compared with the gentamicin group. GM indicates gentamicin.

Article Snippet: To test whether inhibition of p53 attenuated gentamicin-mediated ferroptosis, cells were first treated with or without a p53 inhibitor pifithrin-α (PFT-α; GlpBio, Shanghai, China; 10 μM) for 24 hours, then with 20 mmol/l gentamicin for 24 hours.

Techniques: Expressing, Western Blot

Journal: Breast Cancer Research

Article Title: Proteomic identification of heat shock protein 90 as a candidate target for p53 mutation reactivation by PRIMA-1 in breast cancer cells

doi: 10.1186/bcr1290

Figure Lengend Snippet: Inhibition of PRIMA-1 mediated transcriptional reactivation function of p53 with pifithrin-α (PFTα). MCF-7 (p53 +/+ ) and GI-101A (mut p53) cells were treated with 100 μM PRIMA-1 for 2, 4 and 8 hours (lanes 1, 2 and 3, respectively). Cells were treated with 20 μM PFTα for 6 hours (lane 4) or with 20 μM PFTα for 2 hours followed by PRIMA-1 for 4 hours (lane 5). 20 μg of protein samples of cell lysates were separated by SDS-PAGE (4 to 20% polyacrylamide) and subjected to Western blot analysis with p53 and p21 primary antibodies. The reactive bands were revealed and detected with the Odyssey™ Infrared Imaging System. β-Actin was used as a loading control for protein samples.

Article Snippet: PRIMA-1 (NSC-281668) was obtained from the Drug Synthesis and Chemistry Branch, National Cancer Institute (Bethesda, MD); pifithrin-α (PFTα) and doxorubicin (adriamycin hydrochloride) were purchased from Biomol Research Inc. (Plymouth Meeting, PA).

Techniques: Inhibition, SDS Page, Western Blot, Imaging, Control

Journal: Cell Death & Disease

Article Title: Degradation of mutant p53H175 protein by Zn(II) through autophagy

doi: 10.1038/cddis.2014.217

Figure Lengend Snippet: Zn(II)-curc compound triggers mutp53H175 protein degradation. ( a ) Stability of endogenous p53H175 protein in SKBR3 and HCT116 cells treated with Zn(II)-curc (100 μ M) for 4–8–16 h. Protein levels were measured with western blot using antibody to p53 (FL393). A shorter and longer exposure of p53 blot is showing. Anti- β -actin was used as protein loading control. ( b ) H1299 cells were transiently transfected with p53H175 plasmid (0.1 μ g) and 24 h after transfection treated with Zn(II)-curc (100 μ M) for 8–16–24 h to monitor p53 stability. Protein levels were measured with western blot using antibody to p53. Anti- β -actin was used as protein loading control. ( c ) SKBR3 cells were left untreated or treated with Zn(II)-curc (100 μ M) for 16 and 24 h before analysis of p53 expression using RT-PCR. β -actin was used as a control for efficiency of RNA extraction and transcription. ( d ) H1299 cells were transiently transfected with p53H175 plasmid (0.1 μ g). Twenty-four hours after transfection cells were treated with Zn(II)-curc (100 μ M) for 8 h. Untreated or Zn(II)-curc-treated cells were then incubated with the translation inhibitor CHX (40 μ M) for the indicated hours to assess p53 protein levels using western immunoblot as in ( a and b ). Lower panel: p53 bands were quantitated using densitometry and plotted as relative amount of p53 levels after ZN(II)-curc treatment compared with control

Article Snippet: The following reagents were used: Zn(II)-curc dissolved in DMSO and used at 100 μ M; the early inhibitor of autophagosome formation 3-MA was dissolved in dH 2 O and used at 5 mM; the inhibitor of autophagic protein degradation CQ was dissolved in dH 2 O and used at 25 μ M; the proteasome inhibitor MG132 was dissolved in DMSO and used at 20 μ M; the p53 inhibitor PFT- α (ENZO Life Sciences, Lausen, Switzerland) was dissolved in DMSO and used at 30 μ M; cisplatin (Teva, Pharma Italia, Italy) was used at 5 μ g/ml and CHX, the inhibitor of protein synthesis was dissolved in dH 2 O and diluted into the medium to a final concentration of 40 μ g/ml.

Techniques: Western Blot, Transfection, Plasmid Preparation, Expressing, Reverse Transcription Polymerase Chain Reaction, RNA Extraction, Incubation

Journal: Cell Death & Disease

Article Title: Degradation of mutant p53H175 protein by Zn(II) through autophagy

doi: 10.1038/cddis.2014.217

Figure Lengend Snippet: Reactivation of wtp53 transactivation by Zn(II)-curc is mechanistically involved in mutp53H175 degradation. ( a ) Stability of endogenous p53H175 protein in SKBR3 cells treated with Zn(II)-curc (100 μ M) for 24 h with or without p53 inhibitor PFT- α (30 μ M). Protein levels were measured with western blot using antibody to p53. Anti- β -actin was used as protein loading control. ( b ) SKBR3 cells were depleted of MDM2 function by siRNA interference, as monitored using RT-PCR analysis (left panel) and 36 h after transfection treated with Zn(II)-curc (100 μ M) for 16 h or left untreated. p53 protein levels (right panel) were measured using western blot as in a . Anti- β -actin was used as protein loading control

Article Snippet: The following reagents were used: Zn(II)-curc dissolved in DMSO and used at 100 μ M; the early inhibitor of autophagosome formation 3-MA was dissolved in dH 2 O and used at 5 mM; the inhibitor of autophagic protein degradation CQ was dissolved in dH 2 O and used at 25 μ M; the proteasome inhibitor MG132 was dissolved in DMSO and used at 20 μ M; the p53 inhibitor PFT- α (ENZO Life Sciences, Lausen, Switzerland) was dissolved in DMSO and used at 30 μ M; cisplatin (Teva, Pharma Italia, Italy) was used at 5 μ g/ml and CHX, the inhibitor of protein synthesis was dissolved in dH 2 O and diluted into the medium to a final concentration of 40 μ g/ml.

Techniques: Western Blot, Reverse Transcription Polymerase Chain Reaction, Transfection

Journal: Cell Death & Disease

Article Title: Degradation of mutant p53H175 protein by Zn(II) through autophagy

doi: 10.1038/cddis.2014.217

Figure Lengend Snippet: Zn(II)-curc triggered p53-dependent autophagy. ( a ) RKO and SKBR3 cells were treated with cisplatin (cispl, 5 μ g/ml) for 16 h, while H1299 cells were transfected with wtp53 plasmid (0.1 μ g). After treatment and transfection, cells were collected, RNA was extracted and DRAM mRNA was monitored using RT-PCR analysis. β -actin mRNA was used as internal control. ( b ) SKBR3 cells (6 × 10 6 ) were plated in 150-mm dish and the day after treated with Zn(II)-curc (100 μ M) for 16 h before being assayed for ChIP with anti-p53 and anti-p73 antibodies. PCR analyses were performed on the immunoprecipitated DNA using primers specific for DRAM gene promoter. A sample representing linear amplification of the total chromatin (input) was included as control. Additional controls included immunoprecipitation performed with nonspecific immunoglobulins (no Ab). ( c ) SKBR3 cells were treated with Zn(II)-curc (100 μ M) for 16 and 24 h with or without co-treatment with autophagy inhibitor CQ (25 μ M) for 24 h before being assayed for RT-PCR of DRAM and MDR1. ( d ) H1299 cells were transiently transfected with p53H175 plasmid (0.1 μ g). Twenty-four hours after transfection, cells were treated with Zn(II)-curc (100 μ M) for 24 h before assayed for RT-PCR of DRAM and MDR1. β -actin was used as a control for efficiency of RNA extraction and transcription. ( e ) SKBR3 cells were treated with Zn(II)-curc (100 μ M) 24 h with or without co-treatment with autophagy inhibitor CQ (25 μ M) before being assayed for RT-PCR of DRAM and MDR1. ( f ) SKBR3 cells treated with Zn(II)-curc (100 μ M) over a 24-h time course with or without p53 inhibitor PFT- α (30 μ M). Protein levels were measured with western blot using antibodies to p53, LC3-II and p21 ( h ). Anti- β -actin was used as protein loading control. ( g ) SKBR3 cells were transfected with control or sip53 vector and 36 h after transfection treated with Zn(II)-curc (100 μ M) for 16 and 24 h. Protein levels were analyses with western blot using antibodies to p53 and LC3-II. Anti- β -actin was used as protein loading control. P53 and LC3-II levels were measured using densitometric analysis of the autoradiographs and plotted as p53/ β -actin and LC3/ β -actin ratios, ±S.D.

Article Snippet: The following reagents were used: Zn(II)-curc dissolved in DMSO and used at 100 μ M; the early inhibitor of autophagosome formation 3-MA was dissolved in dH 2 O and used at 5 mM; the inhibitor of autophagic protein degradation CQ was dissolved in dH 2 O and used at 25 μ M; the proteasome inhibitor MG132 was dissolved in DMSO and used at 20 μ M; the p53 inhibitor PFT- α (ENZO Life Sciences, Lausen, Switzerland) was dissolved in DMSO and used at 30 μ M; cisplatin (Teva, Pharma Italia, Italy) was used at 5 μ g/ml and CHX, the inhibitor of protein synthesis was dissolved in dH 2 O and diluted into the medium to a final concentration of 40 μ g/ml.

Techniques: Transfection, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Immunoprecipitation, Amplification, RNA Extraction, Western Blot