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

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: CNS Neuroscience & Therapeutics

Article Title: Pifithrin‐α Enhances the Survival of Transplanted Neural Stem Cells in Stroke Rats by Inhibiting p53 Nuclear Translocation

doi: 10.1111/cns.12045

Figure Lengend Snippet: The protective effect of pifithrin‐α (PFT‐α) on oxygen glucose deprivation (OGD)‐induced neural stem cells (NSCs) injury in vitro. (A) NSCs were exposed to OGD for 0–24 h as indicated. The viability of NSCs was reduced by OGD exposure in a time‐dependent manner (*P < 0.05 and **P < 0.01 vs. control, respectively); the OGD‐induced reduction in viability reached plateau at 12 h. (B) OGD‐induced reduction in cell viability was enhanced by PFT‐α in a dose‐dependent manner (*P < 0.05 and **P < 0.01 vs. control, respectively). (C) Representative images of the neurospheres under the conditions as indicated (scale bar = 100 μm; n = 6 neurospheres in each group). Ctr + vehicle and Ctr + PFT‐α indicate that the neurospheres were cultured under the normal condition for 7 days in the presence of DMSO or PFT‐α. OGD + vehicle and OGD + PFT‐α indicate that the neurospheres were exposed to OGD for 1 h and subsequently were cultured under the normal condition for 7 days in the presence of DMSO or PFT‐α. The diameter of neurospheres was measured on day 7 for each experiment group. (D) The growth of neurospheres was significantly inhibited by OGD exposure; PFT‐α attenuated the inhibitory effect of OGD on neurosphere growth (*P < 0.01 compared with Ctr + vehicle; # P < 0.01 compared with Ctr + PFT‐α; $ P < 0.01 vs. OGD + vehicle). (E, F) PFT‐α attenuates OGD‐induced apoptosis of NSCs. (E) Representative TUNEL staining fields were acquired from different experimental groups as indicated. The NSCs were exposed to OGD for 0, 1, 3, or 6 h in the absence or in the presence of PFT‐α (10 μM). The cells undergoing apoptosis were positively stained with TUNEL reagent (shown in green; scale bar = 20 μm). (F) Quantitative analysis of TUNEL‐positive cells from different groups (n = 3 independent experiments). TUNEL‐positive cells were counted from 10 images obtained in randomly selected fields from each group (##P < 0.01 vs. OGD + vehicle).

Article Snippet: The cell viability of NSCs began to decrease with OGD exposure times of >1 h; therefore, we evaluated the protective effect of PFT‐α (>98%; Beyotime) on NSCs at the time points of 3, 6, and 12 h. A final concentration of 0, 0.5, 1, 5, 10, 20, 40, and 80 μM PFT‐α was added, respectively, into the glucose‐free NBM‐B27 medium under OGD conditions.

Techniques: In Vitro, Control, Cell Culture, TUNEL Assay, Staining

Journal: CNS Neuroscience & Therapeutics

Article Title: Pifithrin‐α Enhances the Survival of Transplanted Neural Stem Cells in Stroke Rats by Inhibiting p53 Nuclear Translocation

doi: 10.1111/cns.12045

Figure Lengend Snippet: (A) Neurological severity scores were evaluated at 2 h before and 1, 3, or 7 days after cell transplantation. Three days after transplantation, the neurological severity scores were declined in the vehicle + neural stem cells (NSCs) and pifithrin‐α (PFT‐α) + NSCs groups at the same degree (*P < 0.05 compared with the control group). On day 7, the neurological severity scores in the PFT‐α + NSCs group were significantly declined compared with those in control group (**P < 0.001) and in the vehicle + NSCs group (# P = 0.024). (B) The differences of neurological severity scores (difference between scores measured on days 1 and 3 or days 1 and 7 after transplantation) were improved significantly in cell‐transplanted rats on days 3 and 7, with further improvement in the PFT‐α + NSCs group on days 7 compared with the vehicle + NSCs group (**P < 0.01 vs. control; # P < 0.05 compared with vehicle + NSCs; n = 6 rats in each group).

Article Snippet: The cell viability of NSCs began to decrease with OGD exposure times of >1 h; therefore, we evaluated the protective effect of PFT‐α (>98%; Beyotime) on NSCs at the time points of 3, 6, and 12 h. A final concentration of 0, 0.5, 1, 5, 10, 20, 40, and 80 μM PFT‐α was added, respectively, into the glucose‐free NBM‐B27 medium under OGD conditions.

Techniques: Transplantation Assay, Control

Journal: CNS Neuroscience & Therapeutics

Article Title: Pifithrin‐α Enhances the Survival of Transplanted Neural Stem Cells in Stroke Rats by Inhibiting p53 Nuclear Translocation

doi: 10.1111/cns.12045

Figure Lengend Snippet: Extent of striatal damage and location of the graft. (A–C) The CT scan images demonstrate the areas of the stroke lesion. The low condensed portions in right hemisphere, where indicated by the white dashed lines, represent the infarction regions. (D) The white dashed line formed circle indicates the graft point of neural stem cells (NSCs; the white arrow head), which is in the vicinity of the stroke‐induced lesion area. (E, F) The grafted NSCs were detected by immunohistochemistry, hematoxylin and eosin staining at the injection site 7 days after transplantation; the brain sections were sliced 20 μm each (×100). (G, H) Seven days after transplantation, six sections (the brain sections were sliced 4 μm each; ×600) from the vehicle + NSCs and pifithrin‐α (PFT‐α) + NSCs groups, respectively, were examined by fluorescence microscopy to observe chloromethylfluorescein diacetate (CMFDA)‐labeled NSCs. The white arrow heads indicate CMFDA‐positive cells. (H) More surviving cells (CMFDA‐positive) were observed in the PFT‐α + NSCs group compared with those in the vehicle + NSC group (16.70 ± 2.69 cells/field vs. 10.55 ± 2.47 cells/field, respectively; **P < 0.01; scale bar = 10 μm).

Article Snippet: The cell viability of NSCs began to decrease with OGD exposure times of >1 h; therefore, we evaluated the protective effect of PFT‐α (>98%; Beyotime) on NSCs at the time points of 3, 6, and 12 h. A final concentration of 0, 0.5, 1, 5, 10, 20, 40, and 80 μM PFT‐α was added, respectively, into the glucose‐free NBM‐B27 medium under OGD conditions.

Techniques: Computed Tomography, Immunohistochemistry, Staining, Injection, Transplantation Assay, Fluorescence, Microscopy, Labeling

Journal: CNS Neuroscience & Therapeutics

Article Title: Pifithrin‐α Enhances the Survival of Transplanted Neural Stem Cells in Stroke Rats by Inhibiting p53 Nuclear Translocation

doi: 10.1111/cns.12045

Figure Lengend Snippet: (A, B) Localization and expression profile of p53 in the grafted neural stem cells (NSCs). Cellular expression and localization of p53 were determined at 12 h after transplantation and viewed by confocal microscopy. In the vehicle + NSCs group, p53 was predominantly localized in nuclei of grafted NSCs; in contrast, p53 was mainly localized in the cytoplasm of grafted NSCs in the pifithrin‐α (PFT‐α) + NSCs group. The images shown in lower panels indicate the amplified fields as indicated by the white frames in the top panels. (C, D) Expression profile of p53‐upregulated modulator of apoptosis (PUMA) in grafted NSCs. (C) Immunofluorescence staining was used to detect the expression of PUMA in the grafted NSCs at 12 h after cell transplantation. The white arrow heads indicate the PUMA‐positive grafted NSCs. Scale bar = 10 μm. (D) The ratio of PUMA‐positive grafted NSCs was significantly reduced in the PFT‐α + NSCs group compared with the vehicle + NSCs group (29.4 ± 5.6% vs. 38.9 ± 4.3%; **P < 0.01).

Article Snippet: The cell viability of NSCs began to decrease with OGD exposure times of >1 h; therefore, we evaluated the protective effect of PFT‐α (>98%; Beyotime) on NSCs at the time points of 3, 6, and 12 h. A final concentration of 0, 0.5, 1, 5, 10, 20, 40, and 80 μM PFT‐α was added, respectively, into the glucose‐free NBM‐B27 medium under OGD conditions.

Techniques: Expressing, Transplantation Assay, Confocal Microscopy, Amplification, Immunofluorescence, Staining