mdm2 smp14  (Santa Cruz Biotechnology)

Journal: Cancer research

Article Title: Peli1 modulates the subcellular localization and activity of Mdmx

doi: 10.1158/0008-5472.CAN-17-3531

Figure Lengend Snippet: (A–B) U2OS Peli1 inducible stable line cells were transfected with control siRNA or siRNA against p53 or Mdmx. After 24 hours, the cells were without treatment or treated with 0.1 μg/ml of doxycycline 48 hours. (A) Western blot analysis of cell extracts with antibody against Peli1, Mdm2, Mdmx, p53, p21 and β-actin. (B) p21 and Mdm2 mRNA levels were determined by real-time PCR in these cells. (C) Western blot analysis of cell extracts from two U2OS Peli1 CRISPR-Cas9 knock-out cell lines (CRISPR 1 and 2) with antibody against Mdm2, Mdmx, p53, Peli1, p21 and β-actin. Parental U2OS cells were used as the control. (D) p21 and Mdm2 mRNA levels were determined by real-time PCR in U2OS Peli1 knock-out cell lines. Parental U2OS cells were used as the control.

Article Snippet: Mouse monoclonal antibody for Peli1 (F-7), p53 (DO-1), Mdm2 (SMP14) and Myc (9E10) and rabbit polyclonal antibodies for p21 (C19) and Puma (H-136) were purchased from Santa Cruz Biotechnology.

Techniques: Transfection, Control, Western Blot, Real-time Polymerase Chain Reaction, CRISPR, Knock-Out

Journal: Molecular medicine reports

Article Title: Matrine‑induced apoptosis in Hep3B cells via the inhibition of MDM2.

doi: 10.3892/mmr.2016.5999

Figure Lengend Snippet: Figure 1. Matrine inhibits the expression of MDM2 in Hep3B and L0‑2 cells. Western blotting was performed to determine (A) the protein expression levels of MDM2 following treatment with 4.0 mg/ml matrine for 24 h, (B) the dose‑response of MDM2 inhibition following treatment with the indicated concentrations of matrine for 24 h, (C) the time‑response of MDM2 inhibition following treatment with 0.5 mg/ml matrine for the indicated durations and (D) the protein expression of MDM2 following treatment with 5 Gy IR for the indicated durations. Western blotting was performed for both in Hep3B and L0‑2 cells, and β‑actin was used as a loading control. Con, control; MDM, mouse double‑minute; IR, irradiation.

Article Snippet: MDM2 CRISPR activation plasmid, and MDM2 (cat. no. BS1223), PUMA (cat. no. AB10418), p73α (cat. no. 4662), p73α (Try99) (cat. no. 4665S) and β-actin (cat. no. 4967) antibodies were all obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Techniques: Expressing, Western Blot, Inhibition, Control, Irradiation

Journal: Molecular medicine reports

Article Title: Matrine‑induced apoptosis in Hep3B cells via the inhibition of MDM2.

doi: 10.3892/mmr.2016.5999

Figure Lengend Snippet: Figure 2. Examination of the molecular mechanism by which matrine reduces MDM2. (A) The mRNA expression of MDM2 in Hep3B cells was determined by RT‑qPCR following treatment with 0.5 mg/ml matrine for the indicated durations. The data is shown as the fold change. (B) Hep3B cells were pre‑treated with or without 10 µM CHX for 10 min, followed by incubation with or without 0.5 mg/ml matrine for 4 h, then 5 mg/ml actino mycin D was added. Cells were then harvested and the mRNA expression levels of MDM2 were determined by RT‑qPCR. The data are presented as the mean ± standard deviation (*P<0.05 and **P<0.01 compared with the untreated 0 h samples). (C) A CHX pulse‑chase assay was performed to detect MDM2 turnover in Hep3B cells treated with or without 0.5 mg/ml matrine for 4 h. The numerical labels under each protein band represents the expression levels after normalization against β‑actin, compared with untreated samples (defined as 1 unit). MDM, mouse double‑minute; CHX, cycloheximide; RT‑qPCR, reverse transcription‑quantitative polymerase chain reaction.

Article Snippet: MDM2 CRISPR activation plasmid, and MDM2 (cat. no. BS1223), PUMA (cat. no. AB10418), p73α (cat. no. 4662), p73α (Try99) (cat. no. 4665S) and β-actin (cat. no. 4967) antibodies were all obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Techniques: Expressing, Incubation, Standard Deviation, Polymerase Chain Reaction

Journal: Molecular medicine reports

Article Title: Matrine‑induced apoptosis in Hep3B cells via the inhibition of MDM2.

doi: 10.3892/mmr.2016.5999

Figure Lengend Snippet: Figure 3. Matrine‑induced downregulation of MDM2 results in an increase in the expression of p73α. (A) Hep3B cells were treated with different concentra tions of either benzo(a)pyrene (16, 32 or 64 µM) or matrine (1.0, 2.0 or 4.0 mg/ml) for 12 h. The mRNA expression levels of p53 were assessed by reverse transcription‑quantitative polymerase chain reaction. The data are presented as the mean ± standard deviation (**P<0.01 compared with the untreated 0 h group) samples. (B) Hep3B cells were pre‑treated with or without 10 µM CHX for 10 min, followed by an incubation with 0.5 mg/ml matrine. The cell extracts from 0 or 2 or 4 h treatment groups were assessed by western blotting to determine the expression of p73α. (C) A pulse‑chase assay for p73α turnover was performed in Hep3B cells treated with 0.5 mg/ml matrine. (D) Hep3B cells treated with 4.0 mg/ml matrine for the indicated durations were assessed for the expression levels of MDM2 and p73α by western blotting. β‑actin was used as a loading control. CHX, cycloheximide; MDM, mouse double‑minute.

Article Snippet: MDM2 CRISPR activation plasmid, and MDM2 (cat. no. BS1223), PUMA (cat. no. AB10418), p73α (cat. no. 4662), p73α (Try99) (cat. no. 4665S) and β-actin (cat. no. 4967) antibodies were all obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Techniques: Expressing, Polymerase Chain Reaction, Standard Deviation, Incubation, Western Blot, Control

Journal: Molecular medicine reports

Article Title: Matrine‑induced apoptosis in Hep3B cells via the inhibition of MDM2.

doi: 10.3892/mmr.2016.5999

Figure Lengend Snippet: Figure 4. Matrine‑increased expression of p73α causes no induction of its targets, p21 and PUMA, as compared with treatment with Benzo(a)pyrene or epoto side. (A) Hep3B cells were treated with 32 µM Benzo(a)pyrene or 0.5 mg/ml matrine for 12 h and subsequently, the mRNA expression levels of p73α, MDM2, p21 and PUMA were assessed by reverse transcription‑quantitative polymerase chain reaction. The data are presented as the fold change compared with the control group. (B) Hep3B cells were treated with either 100 µM epotoside or 0.5 mg/ml matrine for various durations. The protein expression levels of p73α, MDM2, p21 and PUMA were assessed by western blotting. β‑actin was used as a loading control. (C) Cell‑cycle analysis in Hep3B cells was performed 4 h post‑treatment with 100 µM epotoside or 0.5 mg/ml matrine. MDM, mouse double‑minute; PUMA, p53 upregulated modulator of apoptosis.

Article Snippet: MDM2 CRISPR activation plasmid, and MDM2 (cat. no. BS1223), PUMA (cat. no. AB10418), p73α (cat. no. 4662), p73α (Try99) (cat. no. 4665S) and β-actin (cat. no. 4967) antibodies were all obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Techniques: Expressing, Polymerase Chain Reaction, Control, Western Blot, Cell Cycle Assay

Journal: Molecular medicine reports

Article Title: Matrine‑induced apoptosis in Hep3B cells via the inhibition of MDM2.

doi: 10.3892/mmr.2016.5999

Figure Lengend Snippet: Figure 5. Effect of epotoside‑increased and matrine‑decreased MDM2 on the expression of IAP3. (A) Hep3B cells were incubated with either 100 µM epo toside or 0.5 mg/ml matrine for the indicated duration. The protein expression levels of MDM2 and IAP3 were assessed by western blotting. (B) Normal and MDM2 overexpression Hep3B cells were treated with 0.5 mg/ml matrine for the indicated durations. The transfection efficiency of MDM2 and IAP3 in MDM2‑overexpressing Hep3B cells were evaluated by western blotting. (C) The protein stability of IAP3 was detected using a pulse‑chase assay following treatment with 0.5 mg/ml matrine. β‑actin was used as a loading control. MDM, mouse double‑minute; IAP, inhibitor of apoptosis protein; CHX, cycloheximide.

Article Snippet: MDM2 CRISPR activation plasmid, and MDM2 (cat. no. BS1223), PUMA (cat. no. AB10418), p73α (cat. no. 4662), p73α (Try99) (cat. no. 4665S) and β-actin (cat. no. 4967) antibodies were all obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Techniques: Expressing, Incubation, Western Blot, Over Expression, Transfection, Control

Journal: Molecular medicine reports

Article Title: Matrine‑induced apoptosis in Hep3B cells via the inhibition of MDM2.

doi: 10.3892/mmr.2016.5999

Figure Lengend Snippet: Figure 1. Matrine inhibits the expression of MDM2 in Hep3B and L0‑2 cells. Western blotting was performed to determine (A) the protein expression levels of MDM2 following treatment with 4.0 mg/ml matrine for 24 h, (B) the dose‑response of MDM2 inhibition following treatment with the indicated concentrations of matrine for 24 h, (C) the time‑response of MDM2 inhibition following treatment with 0.5 mg/ml matrine for the indicated durations and (D) the protein expression of MDM2 following treatment with 5 Gy IR for the indicated durations. Western blotting was performed for both in Hep3B and L0‑2 cells, and β‑actin was used as a loading control. Con, control; MDM, mouse double‑minute; IR, irradiation.

Article Snippet: MDM2 CRISPR activation plasmid, and MDM2 (cat. no. BS1223), PUMA (cat. no. AB10418), p73α (cat. no. 4662), p73α (Try99) (cat. no. 4665S) and β-actin (cat. no. 4967) antibodies were all obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Techniques: Expressing, Western Blot, Inhibition, Control, Irradiation

Journal: Molecular medicine reports

Article Title: Matrine‑induced apoptosis in Hep3B cells via the inhibition of MDM2.

doi: 10.3892/mmr.2016.5999

Figure Lengend Snippet: Figure 2. Examination of the molecular mechanism by which matrine reduces MDM2. (A) The mRNA expression of MDM2 in Hep3B cells was determined by RT‑qPCR following treatment with 0.5 mg/ml matrine for the indicated durations. The data is shown as the fold change. (B) Hep3B cells were pre‑treated with or without 10 µM CHX for 10 min, followed by incubation with or without 0.5 mg/ml matrine for 4 h, then 5 mg/ml actino mycin D was added. Cells were then harvested and the mRNA expression levels of MDM2 were determined by RT‑qPCR. The data are presented as the mean ± standard deviation (*P<0.05 and **P<0.01 compared with the untreated 0 h samples). (C) A CHX pulse‑chase assay was performed to detect MDM2 turnover in Hep3B cells treated with or without 0.5 mg/ml matrine for 4 h. The numerical labels under each protein band represents the expression levels after normalization against β‑actin, compared with untreated samples (defined as 1 unit). MDM, mouse double‑minute; CHX, cycloheximide; RT‑qPCR, reverse transcription‑quantitative polymerase chain reaction.

Article Snippet: MDM2 CRISPR activation plasmid, and MDM2 (cat. no. BS1223), PUMA (cat. no. AB10418), p73α (cat. no. 4662), p73α (Try99) (cat. no. 4665S) and β-actin (cat. no. 4967) antibodies were all obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Techniques: Expressing, Incubation, Standard Deviation, Polymerase Chain Reaction

Journal: Molecular medicine reports

Article Title: Matrine‑induced apoptosis in Hep3B cells via the inhibition of MDM2.

doi: 10.3892/mmr.2016.5999

Figure Lengend Snippet: Figure 3. Matrine‑induced downregulation of MDM2 results in an increase in the expression of p73α. (A) Hep3B cells were treated with different concentra tions of either benzo(a)pyrene (16, 32 or 64 µM) or matrine (1.0, 2.0 or 4.0 mg/ml) for 12 h. The mRNA expression levels of p53 were assessed by reverse transcription‑quantitative polymerase chain reaction. The data are presented as the mean ± standard deviation (**P<0.01 compared with the untreated 0 h group) samples. (B) Hep3B cells were pre‑treated with or without 10 µM CHX for 10 min, followed by an incubation with 0.5 mg/ml matrine. The cell extracts from 0 or 2 or 4 h treatment groups were assessed by western blotting to determine the expression of p73α. (C) A pulse‑chase assay for p73α turnover was performed in Hep3B cells treated with 0.5 mg/ml matrine. (D) Hep3B cells treated with 4.0 mg/ml matrine for the indicated durations were assessed for the expression levels of MDM2 and p73α by western blotting. β‑actin was used as a loading control. CHX, cycloheximide; MDM, mouse double‑minute.

Article Snippet: MDM2 CRISPR activation plasmid, and MDM2 (cat. no. BS1223), PUMA (cat. no. AB10418), p73α (cat. no. 4662), p73α (Try99) (cat. no. 4665S) and β-actin (cat. no. 4967) antibodies were all obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Techniques: Expressing, Polymerase Chain Reaction, Standard Deviation, Incubation, Western Blot, Control

Journal: Molecular medicine reports

Article Title: Matrine‑induced apoptosis in Hep3B cells via the inhibition of MDM2.

doi: 10.3892/mmr.2016.5999

Figure Lengend Snippet: Figure 4. Matrine‑increased expression of p73α causes no induction of its targets, p21 and PUMA, as compared with treatment with Benzo(a)pyrene or epoto side. (A) Hep3B cells were treated with 32 µM Benzo(a)pyrene or 0.5 mg/ml matrine for 12 h and subsequently, the mRNA expression levels of p73α, MDM2, p21 and PUMA were assessed by reverse transcription‑quantitative polymerase chain reaction. The data are presented as the fold change compared with the control group. (B) Hep3B cells were treated with either 100 µM epotoside or 0.5 mg/ml matrine for various durations. The protein expression levels of p73α, MDM2, p21 and PUMA were assessed by western blotting. β‑actin was used as a loading control. (C) Cell‑cycle analysis in Hep3B cells was performed 4 h post‑treatment with 100 µM epotoside or 0.5 mg/ml matrine. MDM, mouse double‑minute; PUMA, p53 upregulated modulator of apoptosis.

Article Snippet: MDM2 CRISPR activation plasmid, and MDM2 (cat. no. BS1223), PUMA (cat. no. AB10418), p73α (cat. no. 4662), p73α (Try99) (cat. no. 4665S) and β-actin (cat. no. 4967) antibodies were all obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Techniques: Expressing, Polymerase Chain Reaction, Control, Western Blot, Cell Cycle Assay

Journal: Molecular medicine reports

Article Title: Matrine‑induced apoptosis in Hep3B cells via the inhibition of MDM2.

doi: 10.3892/mmr.2016.5999

Figure Lengend Snippet: Figure 5. Effect of epotoside‑increased and matrine‑decreased MDM2 on the expression of IAP3. (A) Hep3B cells were incubated with either 100 µM epo toside or 0.5 mg/ml matrine for the indicated duration. The protein expression levels of MDM2 and IAP3 were assessed by western blotting. (B) Normal and MDM2 overexpression Hep3B cells were treated with 0.5 mg/ml matrine for the indicated durations. The transfection efficiency of MDM2 and IAP3 in MDM2‑overexpressing Hep3B cells were evaluated by western blotting. (C) The protein stability of IAP3 was detected using a pulse‑chase assay following treatment with 0.5 mg/ml matrine. β‑actin was used as a loading control. MDM, mouse double‑minute; IAP, inhibitor of apoptosis protein; CHX, cycloheximide.

Article Snippet: MDM2 CRISPR activation plasmid, and MDM2 (cat. no. BS1223), PUMA (cat. no. AB10418), p73α (cat. no. 4662), p73α (Try99) (cat. no. 4665S) and β-actin (cat. no. 4967) antibodies were all obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Techniques: Expressing, Incubation, Western Blot, Over Expression, Transfection, Control