p53 wild type human osteosarcoma u2os  (ATCC)

Journal: FEBS Open Bio

Article Title: Histone deacetylase 2 is involved in DNA damage‐mediated cell death of human osteosarcoma cells through stimulation of the ATM /p53 pathway

doi: 10.1002/2211-5463.12585

Figure Lengend Snippet: U2 OS cells undergo cell death in response to ADR . (A) WST assay. U2 OS cells were treated with the indicated concentrations of ADR . Twenty‐four hours after treatment, cell viability was assessed by WST assay. (B) FACS analysis. U2 OS cells were exposed to the increasing concentrations of ADR . Twenty‐four hours after treatment, floating and attached cells were collected and analyzed by FACS . Representative microscopic images of these cells are indicated. (C) Trypan blue assay. U2 OS cells were treated as in (A). Twenty‐four hours after treatment, floating and attached cells were harvested, mixed with 0.4% trypan blue solution and number of trypan blue‐positive cells (dead cells) was measured. (D) Immunoblotting. U2 OS cells were treated as in (A). Twenty‐four hours after treatment, cell lysates were extracted and analyzed by immunoblotting. Actin was used as a loading control. (E) Indirect immunostaining. U2 OS cells were exposed to ADR (0.5 μ m ). Twenty‐four hours after treatment, cells were fixed and simultaneously stained with anti‐p53 and anti‐ HDAC 2 antibodies. Cell nuclei were stained with 4′,6‐diamidino‐2‐phenylindole ( DAPI ). (F) RT ‐ PCR . U2 OS cells were treated as in (A). Twenty‐four hours after treatment, total RNA was prepared and subjected to RT ‐ PCR . GAPDH was used as an internal control. All results shown are representative of at least three independent experiments. The error bars represent SD .

Article Snippet: p53 ‐wild‐type human osteosarcoma U2OS and p53 ‐null human lung carcinoma H1299 cells were obtained from ATCC (Manassas, VA, USA), and maintained in Dulbecco's modified Eagle's medium supplemented with 10% heat‐inactivated FBS.

Techniques: WST Assay, Western Blot, Control, Immunostaining, Staining, Reverse Transcription Polymerase Chain Reaction

Journal: FEBS Open Bio

Article Title: Histone deacetylase 2 is involved in DNA damage‐mediated cell death of human osteosarcoma cells through stimulation of the ATM /p53 pathway

doi: 10.1002/2211-5463.12585

Figure Lengend Snippet: si RNA ‐mediated p53 knockdown attenuates ADR ‐induced cell death in U2 OS cells. (A) Western blot analysis. U2 OS cells were transfected with control si RNA or with si RNA against p53 . Twenty‐four hours after transfection, cells were treated with ADR (0.5 μ m ) or left untreated. Expression of p53 was detected by the indicated antibody. (B) Representative microscopic images of U2 OS cells treated as in (A). (C) Trypan blue assay. U2 OS cells were transfected and treated as in (A). Twenty‐four hours after treatment, floating and attached cells were harvested and subjected to trypan blue assay. All results represent at least three independent experiments. Data show mean ± SD ( n = 3, P < 0.05). Data were compared using one‐way ANOVA .

Article Snippet: p53 ‐wild‐type human osteosarcoma U2OS and p53 ‐null human lung carcinoma H1299 cells were obtained from ATCC (Manassas, VA, USA), and maintained in Dulbecco's modified Eagle's medium supplemented with 10% heat‐inactivated FBS.

Techniques: Knockdown, Western Blot, Transfection, Control, Expressing

Journal: FEBS Open Bio

Article Title: Histone deacetylase 2 is involved in DNA damage‐mediated cell death of human osteosarcoma cells through stimulation of the ATM /p53 pathway

doi: 10.1002/2211-5463.12585

Figure Lengend Snippet: Depletion of HDAC 2 impairs ADR ‐dependent activation of p53. (A) Immunoblotting. U2 OS cells were transfected with control siRNA or with siRNA targeting HDAC2. Twenty‐four hours after transfection, cells were treated with ADR (0.5 μ m ) or left untreated. Twenty‐four hours after treatment, cell lysates were prepared and analyzed by immunoblotting. The ratios of p53, p‐p53 at Ser‐15 and Ace‐p53 at Lys‐373/382 in knocked down cells to total p53 in control cells as examined by densitometric analysis are also shown. (B) Indirect immunostaining. HDAC 2 ‐knocked down U2 OS cells were exposed to ADR (0.5 μ m ). Twenty‐four hours after treatment, cells were simultaneously stained with anti‐ HDAC 2 and anti‐p53 antibodies. Cell nuclei were stained with DAPI . (C) RT ‐ PCR . U2 OS cells were transfected and treated as in (A). Twenty‐four hours after treatment, total RNA was extracted and analyzed by RT ‐ PCR . (D) Immunoprecipitation assay. U2 OS cells were exposed to ADR (0.5 μ m ). Twenty‐four hours after treatment, cell lysates were immunoprecipitated with normal mouse serum or with monoclonal anti‐ ATM antibody. The resultant immunoprecipitates were analyzed by immunoblotting with the indicated antibodies (lower); 1/20 of inputs are also shown (upper). All results shown are representative of at least three independent experiments.

Article Snippet: p53 ‐wild‐type human osteosarcoma U2OS and p53 ‐null human lung carcinoma H1299 cells were obtained from ATCC (Manassas, VA, USA), and maintained in Dulbecco's modified Eagle's medium supplemented with 10% heat‐inactivated FBS.

Techniques: Activation Assay, Western Blot, Transfection, Control, Immunostaining, Staining, Reverse Transcription Polymerase Chain Reaction, Immunoprecipitation

Journal: FEBS Open Bio

Article Title: Histone deacetylase 2 is involved in DNA damage‐mediated cell death of human osteosarcoma cells through stimulation of the ATM /p53 pathway

doi: 10.1002/2211-5463.12585

Figure Lengend Snippet: Cell death‐potentiating effect of HDAC 2 depends on p53. H1299 cells were transfected with the indicated combinations of the expression plasmids. Twenty‐four hours after transfection, cells were treated with or without 0.5 μ m of ADR . Twenty‐four hours after treatment, floating and attached cells were harvested and analyzed by trypan blue assay. All results shown are representative of at least three independent experiments. Data show mean ± SD ( n = 3, P < 0.05 or P < 0.01). Data were compared using one‐way ANOVA .

Article Snippet: p53 ‐wild‐type human osteosarcoma U2OS and p53 ‐null human lung carcinoma H1299 cells were obtained from ATCC (Manassas, VA, USA), and maintained in Dulbecco's modified Eagle's medium supplemented with 10% heat‐inactivated FBS.

Techniques: Transfection, Expressing

Journal: FEBS Open Bio

Article Title: Histone deacetylase 2 is involved in DNA damage‐mediated cell death of human osteosarcoma cells through stimulation of the ATM /p53 pathway

doi: 10.1002/2211-5463.12585

Figure Lengend Snippet: HDAC 2 increases ADR ‐induced cell death and enhances the transcriptional activity of p53. (A) Forced expression of HA ‐ HDAC 2. U2 OS cells were transfected with the empty plasmid or with the expression plasmid for HA ‐ HDAC 2. Forty‐eight hours after transfection, cell lysates were analyzed by immunoblotting with anti‐ HDAC 2 (upper) or with anti‐actin (lower) antibody. (B) Representative microscopic images of HDAC 2‐overexpressed U2 OS cells in response to ADR . U2 OS cells were transfected with the empty plasmid or with the expression plasmid for HA ‐ HDAC 2. Twenty‐four hours after transfection, cells were exposed to ADR (0.5 μ m ). Twenty‐four hours after treatment, photos were taken. (C) Trypan blue assay. Floating and attached cells in (B) were harvested and mixed with 0.4% trypan blue solution and the number of trypan blue‐positive cells (dead cells) was measured. All results shown are representative of at least three independent experiments. Data show mean ± SD ( n = 3, P < 0.05). (D) Luciferase reporter assay. H1299 cells were transfected with the luciferase construct bearing p21 WAF 1 or NOXA promoter, Renilla luciferase plasmid and a constant amount of p53 expression plasmid plus increasing amounts of HA ‐ HDAC 2 expression plasmid. Forty‐eight hours after transfection, cell lysates were prepared and their luciferase activities driven by p21 WAF 1 ( n = 3, P < 0.01) or NOXA ( n = 3, P < 0.05) promoter were measured. All results shown are representative of at least three independent experiments. Data were compared using one‐way ANOVA .

Article Snippet: p53 ‐wild‐type human osteosarcoma U2OS and p53 ‐null human lung carcinoma H1299 cells were obtained from ATCC (Manassas, VA, USA), and maintained in Dulbecco's modified Eagle's medium supplemented with 10% heat‐inactivated FBS.

Techniques: Activity Assay, Expressing, Transfection, Plasmid Preparation, Western Blot, Luciferase, Reporter Assay, Construct

Journal: Current Research in Structural Biology

Article Title: Comparative computational and experimental analyses of some natural small molecules to restore transcriptional activation function of p53 in cancer cells harbouring wild type and p53 Ser46 mutant

doi: 10.1016/j.crstbi.2022.09.002

Figure Lengend Snippet: Summary of the structures downloaded from PDB and PubChem along with their identification number.

Article Snippet: Human osteosarcoma (U2OS; wild type p53) and oral squamous carcinoma (HSC3; p53 Ser46mutant ) cells were obtained from the Japanese Collection of Research Bioresources (JCRB, Tokyo, Japan).

Techniques:

Journal: Current Research in Structural Biology

Article Title: Comparative computational and experimental analyses of some natural small molecules to restore transcriptional activation function of p53 in cancer cells harbouring wild type and p53 Ser46 mutant

doi: 10.1016/j.crstbi.2022.09.002

Figure Lengend Snippet: Docking scores of the ligands which bound with mutated p53/p62 complexes.

Article Snippet: Human osteosarcoma (U2OS; wild type p53) and oral squamous carcinoma (HSC3; p53 Ser46mutant ) cells were obtained from the Japanese Collection of Research Bioresources (JCRB, Tokyo, Japan).

Techniques:

Journal: Current Research in Structural Biology

Article Title: Comparative computational and experimental analyses of some natural small molecules to restore transcriptional activation function of p53 in cancer cells harbouring wild type and p53 Ser46 mutant

doi: 10.1016/j.crstbi.2022.09.002

Figure Lengend Snippet: Molecular dynamic simulation study of natural metabolites on phosphor mutant p53-p62 complex. A) Conformational changes observed in the structure of mutant type p53 compared with wild-type p53. B) Conformational changes observed in the structure of mutant type p53 after Cuc-B interaction. C) Conformational changes observed in the structure of mutant type p53 after Wi-N interaction. D) Conformational changes observed in the structure of mutant type p53 after Wi-A interaction. E) Root mean square deviation plot of wild-type as well as mutant p53 complex with p62 before and after Cuc-B interaction simulated for 200ns. F) Root mean square deviation plot of mutant p53 complex with p62 before and after Wi-N, Withaferin-A, CAPE and ARC interaction simulated for 200ns. G) Interaction fraction diagram of wild type p53 with amino acids of p62. H) Interaction fraction diagram of phospho-mutant p53 with amino acids of p62.

Article Snippet: Human osteosarcoma (U2OS; wild type p53) and oral squamous carcinoma (HSC3; p53 Ser46mutant ) cells were obtained from the Japanese Collection of Research Bioresources (JCRB, Tokyo, Japan).

Techniques: Mutagenesis

Journal: Current Research in Structural Biology

Article Title: Comparative computational and experimental analyses of some natural small molecules to restore transcriptional activation function of p53 in cancer cells harbouring wild type and p53 Ser46 mutant

doi: 10.1016/j.crstbi.2022.09.002

Figure Lengend Snippet: Molecular dynamics simulation analysis of natural metabolites in restoring wild type p53 activity. A) Interaction fraction diagram of phospho-mutant p53 with amino acids of p62 after Cuc-B intervention. B) Interaction fraction diagram of phospho-mutant p53 with amino acids of p62 after Wi-N intervention. C) Interaction fraction diagram of phospho-mutant p53 with amino acids of p62 after Wi-A intervention. D) Change in MMMGBSA binding energy before and after Cuc-B interaction. E) Change in MMMGBSA binding energy before and after the Wi-N interaction. F) Change in MMMGBSA binding energy before and after the Wi-A interaction. G) Change in Vander Wal, electrostatic and hydrogen bonding energy of interacting molecules. H) Hydrogen bonding plot observed between p53 and p62 before and after Cuc-B intervention.

Article Snippet: Human osteosarcoma (U2OS; wild type p53) and oral squamous carcinoma (HSC3; p53 Ser46mutant ) cells were obtained from the Japanese Collection of Research Bioresources (JCRB, Tokyo, Japan).

Techniques: Activity Assay, Mutagenesis, Binding Assay

Journal: Current Research in Structural Biology

Article Title: Comparative computational and experimental analyses of some natural small molecules to restore transcriptional activation function of p53 in cancer cells harbouring wild type and p53 Ser46 mutant

doi: 10.1016/j.crstbi.2022.09.002

Figure Lengend Snippet: Molecular dynamics simulation study of Mortalin p53 abrogation by natural metabolites. A) Three-dimensional visualization of interaction between Mortalin and p53 (docked using HADDOCK server and simulated for 200ns). B) Three-dimensional visualization of Interaction between Cuc-B and p53 binding domain of Mortalin. C) Three-dimensional visualization of Interaction between Wi-N and p53 binding domain of Mortalin. D) Three-dimensional visualization of interaction between Wi-A and p53 binding domain of Mortalin. E) Three-dimensional visualization of interaction between CAPE and p53 binding domain of Mortalin. F) Three-dimensional visualization of Interaction between Cuc-B and Mortalin binding domain of p53. G) Root mean square deviation plot of natural ligands bound Mortalin complex showing stable interactions. H) Root mean square deviation plot of Cuc-B bound p53 complex showing stable interaction.

Article Snippet: Human osteosarcoma (U2OS; wild type p53) and oral squamous carcinoma (HSC3; p53 Ser46mutant ) cells were obtained from the Japanese Collection of Research Bioresources (JCRB, Tokyo, Japan).

Techniques: Binding Assay

Journal: Current Research in Structural Biology

Article Title: Comparative computational and experimental analyses of some natural small molecules to restore transcriptional activation function of p53 in cancer cells harbouring wild type and p53 Ser46 mutant

doi: 10.1016/j.crstbi.2022.09.002

Figure Lengend Snippet: Dock scores and MMGBSA binding energy for the interaction of the ligands with p53 and Mortalin.

Article Snippet: Human osteosarcoma (U2OS; wild type p53) and oral squamous carcinoma (HSC3; p53 Ser46mutant ) cells were obtained from the Japanese Collection of Research Bioresources (JCRB, Tokyo, Japan).

Techniques: Binding Assay

Journal: Current Research in Structural Biology

Article Title: Comparative computational and experimental analyses of some natural small molecules to restore transcriptional activation function of p53 in cancer cells harbouring wild type and p53 Ser46 mutant

doi: 10.1016/j.crstbi.2022.09.002

Figure Lengend Snippet: Interacting residues of p53 and Mortalin with the ligands after simulation for 200ns.

Article Snippet: Human osteosarcoma (U2OS; wild type p53) and oral squamous carcinoma (HSC3; p53 Ser46mutant ) cells were obtained from the Japanese Collection of Research Bioresources (JCRB, Tokyo, Japan).

Techniques:

Journal: Current Research in Structural Biology

Article Title: Comparative computational and experimental analyses of some natural small molecules to restore transcriptional activation function of p53 in cancer cells harbouring wild type and p53 Ser46 mutant

doi: 10.1016/j.crstbi.2022.09.002

Figure Lengend Snippet: In vitro analysis of the comparative wild type p53 activation function of five natural compounds. A) Western blotting of control and treated U2OS cells for p53 and p21 proteins showed increase in the treated cells. Highest increase was observed in cells treated with Withaferin-A followed by Withanone, Cucurbitacin B, CAPE and Artepillin C. B) Immunocytostaining of control and treated U2OS cells showed increase in expression of p53 and p21 in the later. C) p53-dependent luciferase reporter assay in control and treated U2OS cells showed increase in wild type p53 activity on treated cells and was in accordance to the expression analysis. D) Western blotting of control and treated HSC3 cells (harboring p53 Ser46mutant ) for p21 showed its increase in the later. Blots (A and D) were probed with β-Actin as an internal loading positive control. The results from three independent experiments are shown with statistical analysis indicated as p values. p ​> ​0.05 (non-significant), p ​≤ ​0.05 (∗significant), p ​≤ ​0.01 (∗∗ very significant), p ≤ 0.001 (∗∗∗highly significant), and p ​≤ ​0.0001 (∗∗∗∗ extremely significant).

Article Snippet: Human osteosarcoma (U2OS; wild type p53) and oral squamous carcinoma (HSC3; p53 Ser46mutant ) cells were obtained from the Japanese Collection of Research Bioresources (JCRB, Tokyo, Japan).

Techniques: In Vitro, Activation Assay, Western Blot, Control, Expressing, Luciferase, Reporter Assay, Activity Assay, Positive Control

Journal: Scientific Reports

Article Title: DBC1/CCAR2 is involved in the stabilization of androgen receptor and the progression of osteosarcoma

doi: 10.1038/srep13144

Figure Lengend Snippet: ( a , b ) The knock-down of DBC1 ( a ) or AR ( b ) with two sets of siRNAs for DBC1 or AR (siDBC1 #1, siDBC1 #2, siAR #1, and siAR #2) inhibit the proliferation of both U2OS and SaOS2 osteosarcoma cells as indicated by an MTT and colony forming assay. ( c ) The knock-down of DBC1 and AR significantly reduced the migration activity of both U2OS and SaOS2 cells. ( d ) The invasion capacity of U2OS and SaOS2 cells are significantly decreased with the knock-down of DBC1 and AR in a matrigel invasion assay. ( e ) Cell cycle analysis with flow cytometry shows that the knock-down of DBC1 significantly increases the subG1 and G0/G1 populations in the both U2OS and SaOS2 cells. The statistical analysis for the cell cycle analysis was performed from the flow-cytometry analysis six times. The MTT assay was performed by seeding 1 × 10 3 U2OS cells and 2 × 10 3 SaOS2 cells, and the absorbance measured at 560 nm. For the colony forming assays, 1 × 10 3 U2OS cells and 2 × 10 3 SaOS2 cells were seeded per well of a 24-well culture plate for seven days. The trans-chamber migration and invasion assays were performed after seeding 4 × 10 4 U2OS cells and 1 × 10 5 SaOS2 cells. * p < 0.05, ** p < 0.001.

Article Snippet: The human osteosarcoma cell lines, U2OS (wild-type P53) and SaOS2 (P53-null) were purchased from the Korean Cell Line Bank (KCLB, Seoul, Korea) and cultured in DMEM medium supplemented with 10% fetal bovine serum (Gibco BRL, Gaithersburg, MD), penicillin and streptomycin (100 U/ml), and fungizone (0.25 μg/ml, Gibco BRL, Gaithersburg, MD) at 37 °C in humidified incubator with 5% CO 2 .

Techniques: Knockdown, Migration, Activity Assay, Invasion Assay, Cell Cycle Assay, Flow Cytometry, MTT Assay

Journal: Scientific Reports

Article Title: DBC1/CCAR2 is involved in the stabilization of androgen receptor and the progression of osteosarcoma

doi: 10.1038/srep13144

Figure Lengend Snippet: ( a , b ) Western blotting indicates that the protein levels of BCL-2, TGFβ, RhoA, NFκB, and PCNA were significantly decreased and the expression of P21 and P27 were significantly increased with the knock-down of DBC1 by siRNA in the both U2OS (wild-type P52) and SaOS2 (P53-null) cells. In U2OS cells, acetylation of P53 was significantly increased with the knock-down of DBC1. Especially, the protein level of AR was significantly decreased with the knock-down of DBC1. ( c ) However, the mRNA level of AR did not decrease with the knock-down of DBC1 as demonstrated by quantitative reverse-transcription PCR. ( d , e ) The knock-down of AR increased the protein levels of P21, P27, and BAX, and decreased the protein levels of TGFβ, RhoA, and NFκB in the both U2OS and SaOS2 cells. But the protein level of DBC1 ( e ) and mRNA level of DBC1 ( f ) were not significantly changed with the knock-down of AR with siRNA for AR. The statistical analysis for the quantification of protein expression was performed from three western blots. * p < 0.05, ** p < 0.001.

Article Snippet: The human osteosarcoma cell lines, U2OS (wild-type P53) and SaOS2 (P53-null) were purchased from the Korean Cell Line Bank (KCLB, Seoul, Korea) and cultured in DMEM medium supplemented with 10% fetal bovine serum (Gibco BRL, Gaithersburg, MD), penicillin and streptomycin (100 U/ml), and fungizone (0.25 μg/ml, Gibco BRL, Gaithersburg, MD) at 37 °C in humidified incubator with 5% CO 2 .

Techniques: Western Blot, Expressing, Knockdown, Reverse Transcription

Journal: Scientific Reports

Article Title: DBC1/CCAR2 is involved in the stabilization of androgen receptor and the progression of osteosarcoma

doi: 10.1038/srep13144

Figure Lengend Snippet: ( a ) DBC1 (green) and AR (red) are co-localized mainly in the nuclei of U2OS and SaOS2 osteosarcoma cells by the confocal microscopic image with immunofluorescence staining. ( b ) Immunoprecipitation indicates direct binding of DBC1 and AR. AR is detected in samples taken with immunoprecipitation for DBC1 and vice versa . ( c ) Transfection of DBC1 siRNA into U2OS cells decreases AR stability via a proteasome-mediated pathway. The protein level of AR was decreased more in cells transfected with DBC1 siRNA than in cells transfected with control siRNA. U2OS cells transfected with control or DBC1 siRNA for 24 hours and treated with DMSO (control vehicle), cycloheximide (CHX, 20 μg/ml), or MG-132 (20 μM) for the indicated time. ( d ) Transfection of DBC1 siRNA into U2OS cells caused ubiquitination of AR. U2OS cells were transfected with control or DBC1 siRNA for 24 hours using Lipofectamine. Subsequently, the transfected cells were treated with MG-132 (20 μM) for 4 h and total lysates of cells were immuno-precipitated with anti-androgen receptor antibodies and blotted with anti-ubiquitin antibodies.

Article Snippet: The human osteosarcoma cell lines, U2OS (wild-type P53) and SaOS2 (P53-null) were purchased from the Korean Cell Line Bank (KCLB, Seoul, Korea) and cultured in DMEM medium supplemented with 10% fetal bovine serum (Gibco BRL, Gaithersburg, MD), penicillin and streptomycin (100 U/ml), and fungizone (0.25 μg/ml, Gibco BRL, Gaithersburg, MD) at 37 °C in humidified incubator with 5% CO 2 .

Techniques: Immunofluorescence, Staining, Immunoprecipitation, Binding Assay, Transfection, Control, Ubiquitin Proteomics

Journal: Cancer Research

Article Title: Phosphorylation and Degradation of MdmX Is Inhibited by Wip1 Phosphatase in the DNA Damage Response

doi: 10.1158/0008-5472.can-09-0634

Figure Lengend Snippet: Figure 1. Wip1 interacts with and dephosphorylates MdmX. A, the effects of overexpressed Ser/Thr protein phosphatases on the levels of MdmX. U2OS cells were transfected with control or phosphatase expression vector, treated by NCS (500 ng/mL), and then harvested 2 h after treatment. Levels of MdmX were detected by immunoblotting and quantitated according to the intensity of MdmX bands. B, Wip1 inhibits the phosphorylation of MdmX. U2OS cells were transfected with control or Wip1 expression vector. Cell lysates were harvested 2 h after NCS (500 ng/mL) and MG132 (25 mmol/L, proteasomal inhibitor) treatment. Protein levels were determined by immunoblotting. C, Wip1 interacts with wild-type and mutant MdmX. Immunoprecipitates (IP) from U2OS cell lysates using control or anti-MdmX antibody were analyzed by immunoblotting using anti-Wip1 antibody (top left). A reciprocal experiment using Wip1-containing immunoprecipitates confirmed the Wip1-MdmX interaction (bottom left). U2OS cells were also transfected with vector DNA expressing wild-type or mutant MdmX with three phosphorylation sites (S342, S367, and S403) mutated to alanines (right). D, Wip1 inhibits the phosphorylation of MdmX and directly dephosphorylates MdmX pSer403 in vitro. Phospho-specific MdmX antibodies were used to measure the effects of Wip1 on the phosphorylation of MdmX (left). Phosphopeptides from p38 MAP kinase [pT180, positive control (pos. ctrl)], UNG2 [pT31, negative control (neg. ctrl)], and MdmX (pS342, pS367, or pS403) were incubated with purified Wip1 proteins in in vitro phosphatase assays. Reactions on MdmX (pSer403) were also performed in the absence of magnesium or peptide, or in the presence of okadaic acid (right).

Article Snippet: U2OS (p53 wild-type) cell line is a human osteosarcoma line that was obtained from the American Type Culture Collection.

Techniques: Transfection, Control, Expressing, Plasmid Preparation, Western Blot, Phospho-proteomics, Mutagenesis, In Vitro, Positive Control, Negative Control, Incubation, Purification

Journal: Cancer Research

Article Title: Phosphorylation and Degradation of MdmX Is Inhibited by Wip1 Phosphatase in the DNA Damage Response

doi: 10.1158/0008-5472.can-09-0634

Figure Lengend Snippet: Figure 2. Wip1 stabilizes MdmX in the DNA damage response. A, MdmX is dephosphorylated and stabilized by Wip1. U2OS cells were transfected with Wip1, Wip1 shRNA, or control expression vector, and then treated with NCS (200 ng/mL). Protein levels were detected by immunoblotting at the indicated time point. B, Wip1+/+ and Wip1/ MEFs were treated with NCS (200 ng/mL) and with or without ATM/ATR inhibitor, CGK733 (4.5 Amol/L). Protein levels were detected by immunoblotting. C, overexpressed Wip1 stabilizes MdmX in the DNA damage response. U2OS cells were transfected with Wip1, Wip1 shRNA, or control expression vector. Cells were treated with cyclohexamide (CHX, 50 Ag/mL) to inhibit protein synthesis. Protein stability of MdmX was measured in cells treated with or without NCS (200 ng/mL). D, levels of MdmX at each time point in C were quantitated and half-life of MdmX was calculated (from two separate experiments).

Article Snippet: U2OS (p53 wild-type) cell line is a human osteosarcoma line that was obtained from the American Type Culture Collection.

Techniques: Transfection, shRNA, Control, Expressing, Plasmid Preparation, Western Blot

Journal: Cancer Research

Article Title: Phosphorylation and Degradation of MdmX Is Inhibited by Wip1 Phosphatase in the DNA Damage Response

doi: 10.1158/0008-5472.can-09-0634

Figure Lengend Snippet: Figure 4. Stabilization of MdmX by Wip1 is dependent on ATM-mediated phosphorylation. A, Wip1 extends the half-lives of wild-type and mutant forms of MdmX. Protein levels of wild-type or mutant forms of MdmX were measured as described in Fig. 2C. Values below the MdmX blots indicate protein levels relative to the time zero point in each set (left). Half-lives of wild-type and mutant forms of MdmX were calculated (right). Columns, mean; bars, SD. B, Wip1 stabilizes MdmX in an ATM-dependent manner. A-T cells (GM9607) were transfected with control or Wip1 expression vector, together with ATM expression or control vector. Cells were then treated with NCS (200 ng/mL), harvested, and immunoblotted as indicated. Levels of MdmX at each time point were quantitated. Values below the MdmX blots indicate protein levels relative to the time zero point under each condition (left). Half-life of MdmX was determined for the graph (right). C, induction of Wip1 has a delayed onset compared with the destabilization of MdmX and Mdm2. U2OS cells were treated with 5 Gy of ionizing radiation and cell lysates were harvested and analyzed by immunoblotting.

Article Snippet: U2OS (p53 wild-type) cell line is a human osteosarcoma line that was obtained from the American Type Culture Collection.

Techniques: Phospho-proteomics, Mutagenesis, Transfection, Control, Expressing, Plasmid Preparation, Western Blot

Journal: Cancer Research

Article Title: Phosphorylation and Degradation of MdmX Is Inhibited by Wip1 Phosphatase in the DNA Damage Response

doi: 10.1158/0008-5472.can-09-0634

Figure Lengend Snippet: Figure 5. Wip1 regulates the activity of p53 in an Mdm2- and MdmX-dependent manner. A, effect of Wip1 on MdmX is dependent on Mdm2. MEFs were transfected with control or Wip1 expression vector, treated with NCS (200 ng/mL) and cyclohexamide (100 Ag/mL), harvested, and immunoblotted as indicated. B, p53 activity shown by p21 induction is inhibited by Wip1 in an Mdm2- and MdmX-dependent manner at the early stage of DNA damage response. MEFs were transfected with p53 expression vector, and control or Wip1 expression vector. Twenty-four hours after transfections, cells were treated with NCS (200 ng/mL), harvested 4 h after treatment, and immunoblotted as indicated. C, MEFs were transfected with p53 expression vector, p21-Luciferase expression vector, Renilla luciferase control vector, along with Wip1 or control expression vector. Cells were treated with NCS (200 ng/mL) or Nutlin-3 (10 Amol/L) as indicated. Eighteen hours after treatment, cells were assayed for relative luciferase activity (versus nontreated cells transfected with control vector DNA in each set) to assess the transcriptional activity of p53. Columns, mean of three independent experiments; bars, SD. Two group comparisons were analyzed by Student’s t test.

Article Snippet: U2OS (p53 wild-type) cell line is a human osteosarcoma line that was obtained from the American Type Culture Collection.

Techniques: Activity Assay, Transfection, Control, Expressing, Plasmid Preparation, Luciferase

Journal: Cancer Research

Article Title: Phosphorylation and Degradation of MdmX Is Inhibited by Wip1 Phosphatase in the DNA Damage Response

doi: 10.1158/0008-5472.can-09-0634

Figure Lengend Snippet: Figure 6. Wip1interfereswith Mdm2-MdmX interaction but enhances USP7-MdmX interaction. A, phospho-mutant forms of MdmX have reduced binding ability with Mdm2 but haveenhancedbinding activity with USP7.293 HEK cellswere transfected with HA-tagged wild-type or mutant MdmX expression vector, treated with NCS (200 ng/mL),and harvested 2 haftertreatment.Celllysateswere immunoprecipitated with anti-Mdm2 or anti-USP7 antibody. MdmX in the immunoprecipitates and various proteins in whole cell lysates (WCL) were assessed by immunoblotting as indicated. WB, Western blotting. B, overexpression of Wip1 inhibits Mdm2-MdmX interaction but promotes USP7-MdmX interaction. 293 HEK cells that express inducible Wip1 with doxycycline were treated with or without NCS (200 ng/mL) and MG132 (25 mmol/L), harvested, and lysed 2 h after treatment. Immunoprecipitation and immunoblotting were performed as described in A. C, Wip1 suppresses the induction of p53 through dephosphorylating and stabilizing Mdm2 and MdmX. Upon DNA damage stress, ATM is activated to phosphorylate MdmX and Mdm2. PhosphorylationofMdmX and Mdm2 expeditestheir destabilization and degradation. p53 is rapidly stabilized and activated to initiate a variety of cell activities. Induced by p53, Wip1 dephosphorylates MdmX and Mdm2, resulting in their stabilization and p53 reduction.

Article Snippet: U2OS (p53 wild-type) cell line is a human osteosarcoma line that was obtained from the American Type Culture Collection.

Techniques: Mutagenesis, Binding Assay, Activity Assay, Transfection, Expressing, Plasmid Preparation, Immunoprecipitation, Western Blot, Over Expression

Journal: Molecular and Cellular Biology

Article Title: The Biological Impact of the Human Master Regulator p53 Can Be Altered by Mutations That Change the Spectrum and Expression of Its Target Genes

doi: 10.1128/mcb.26.6.2297-2308.2006

Figure Lengend Snippet: FIG. 1. Transient transfection of p53 mutants changes biological out- comes (phenotypes). (A) Appearance of p53. Presented is a Western blot analysis of p53 levels found in SaOS2 cell lysates at 24 h after transfection. The p53 proteins were detected with DO-1 and pAb1801 antibodies. Immunodetection of actin was used as a control. (B) Suppression of colony growth. SaOS2 cells were transfected using 1.5 g of pCMV-Neo plasmids containing different p53 alleles under the control of a cytomeg- alovirus promoter and subjected to drug selection. Colonies were counted after 12 to 14 days of selection. (C) Evaluation of apoptosis induction by mutant p53s. Twenty-four hours after transfection, cells were simulta- neously stained with propidium iodide and acridine orange, as described in Materials and Methods. The relative percentages of apoptotic cells were determined based on nuclear morphology and stain pattern. The bar graphs show the averages of three independent experiments and include standard deviations (SD). WT, wild type.

Article Snippet: Human osteosarcoma SaOS2 (p53 null) and U2OS (p53 wild type) cell lines obtained from ATCC were maintained in Dulbecco’s modified Eagle medium or McCoy’s 5A with 10% FBS and antibiotics.

Techniques: Transfection, Western Blot, Immunodetection, Control, Selection, Mutagenesis, Staining

Journal: Molecular and Cellular Biology

Article Title: The Biological Impact of the Human Master Regulator p53 Can Be Altered by Mutations That Change the Spectrum and Expression of Its Target Genes

doi: 10.1128/mcb.26.6.2297-2308.2006

Figure Lengend Snippet: FIG. 2. Inducible regulated expression of p53 mutants results in different levels of apoptosis induction. (A) Western blot analysis of p53 induction. Stable SaOS2 cell lines with the different p53 transgenes regulated by the Tet-Off promoter were cultured in medium containing dox. Lanes 1 to 5 correspond to p53. Lanes 6 to 10 correspond to cells growing for 24 h in medium lacking dox (i.e., p53 ON). Immunodetection of actin was used as an internal control. The numbers following the p53 designations refer to the cell lines. (B) Induction of apoptosis by expressed p53 mutants. SaOS2 cells were harvested 24 h after dox removal from the medium (i.e., p53 ON). The cells were stained with propidium iodide and subjected to flow cytometry. (C) Evaluation of apoptosis by annexin V staining and flow cytometry. SaOS2 inducible cell lines were harvested and counted 24 h after dox removal (p53 ON). Cells (1 105) from each sample were stained with PE-conjugated annexin V and then analyzed by FACS. Presented are the average percentages and SD from three independent experiments. WT, wild type.

Article Snippet: Human osteosarcoma SaOS2 (p53 null) and U2OS (p53 wild type) cell lines obtained from ATCC were maintained in Dulbecco’s modified Eagle medium or McCoy’s 5A with 10% FBS and antibiotics.

Techniques: Expressing, Western Blot, Cell Culture, Immunodetection, Control, Staining, Cytometry

Journal: Molecular and Cellular Biology

Article Title: The Biological Impact of the Human Master Regulator p53 Can Be Altered by Mutations That Change the Spectrum and Expression of Its Target Genes

doi: 10.1128/mcb.26.6.2297-2308.2006

Figure Lengend Snippet: FIG. 3. Expression of p53 mutants affects resistance to UV and radiation. (A) Cell death. SaOS2 cells with p53 mutants under the Tet-inducible promoter were grown in the presence of dox (p53 OFF). Six hours before UV or irradiation, dox was removed (p53 ON). Cells were examined at 24 h after exposure to UV (10 J/m2) or radiation (4 Gy) or no radiation. Total cell death is the sum of early and late apoptosis as well as necrosis quantified by flow cytometry analysis of 7-AAD- and annexin V-PE-stained cells. (B) Clonal survival. SaOS2 cells containing p53 mutants under the Tet-inducible promoter were grown in the presence of dox (p53 OFF) or in the absence of dox (p53 ON), and percent clonal survival was determined 21 days later. (C) Changes in sensitivity to UV resulting from expressed p53 mutants. (D) Changes in sensitivity to radiation resulting from expressed p53 mutants. Clonal survival of radiation-treated cells containing induced p53 was compared to p53 ON cells that were not irradiated. Presented are results and SD from three independent experiments. WT, wild type.

Article Snippet: Human osteosarcoma SaOS2 (p53 null) and U2OS (p53 wild type) cell lines obtained from ATCC were maintained in Dulbecco’s modified Eagle medium or McCoy’s 5A with 10% FBS and antibiotics.

Techniques: Expressing, Irradiation, Cytometry, Staining

Journal: Molecular and Cellular Biology

Article Title: The Biological Impact of the Human Master Regulator p53 Can Be Altered by Mutations That Change the Spectrum and Expression of Its Target Genes

doi: 10.1128/mcb.26.6.2297-2308.2006

Figure Lengend Snippet: FIG. 5. Analysis of transactivation by the wild type and various p53 mutants at p53-responsive promoters. The P21, MDM2-P2, and MHS2 promoters were tested. Plasmids containing luciferase reporter constructs were cotransfected together with p53 expression plasmids and the control vector pRL-SV40 into SaOS2 cells, as described in Materials and Meth- ods. Cell lysates were prepared 48 h after transfection and luciferase activity was measured. Results were normalized using the Renilla consti- tutive luciferase activity. The relative means and the SD for at least three independent experiments are presented. WT, wild type.

Article Snippet: Human osteosarcoma SaOS2 (p53 null) and U2OS (p53 wild type) cell lines obtained from ATCC were maintained in Dulbecco’s modified Eagle medium or McCoy’s 5A with 10% FBS and antibiotics.

Techniques: Luciferase, Construct, Expressing, Control, Plasmid Preparation, Transfection, Activity Assay