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    Name:
    p53 r PR
    Description:
    Gene Silencers generally consist of pools of three to five target specific 19 25 nucleotide sequences in length For independent verification of p53 gene silencing results individual duplex components or plasmids are also available upon request Suitable control antibody p53 Antibody A 1 sc 393031 is recommended as control antibody for monitoring of p53 expression knockdown by Western blotting or immunofluorescence
    Catalog Number:
    SC-45917-PR
    Price:
    None
    Category:
    Gene Editing siRNA shRNA Gene Silencers Tumor Suppressor Apoptosis p53 siRNA shRNA Plasmid and Lentiviral Particle Gene Silencers p53 siRNA and shRNA Plasmids r
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    Structured Review

    Santa Cruz Biotechnology p53
    Dox, AD 312, and AD 198 treatments increased caspase-3/7 activity and cleaved caspase-3 protein expression in <t>wt-p53</t> bladder TCC cells The wt-p53 RT4 and SW780 cells, and the mt-p53 5637, UM-UC-3, T-24, J82, and TCCSUP cells were treated with 1 µM Dox, 10 µM AD 312, and 1 µM AD 198 for 24 hours. ( A ) The activities of caspase 3/7 were measured by Caspase-Glo3/7 luminescence assay. A significant increase in caspase 3/7 activities by Dox, AD 312 and AD 198 treatments were detected in wt-p53 RT4 and SW780 cells. Only moderate upregulation of caspase 3/7 activities by Dox, AD 312 and AD 198 were observed in mt-p53 5637 cells. Dox treatment significantly increased caspase 3/7 activity in mt-p53 UM-UC-3 and T-24 cells. Low caspase 3/7 activities were detected in mt-p53 J82 and TCCSUP cells. Data shown here are mean ± S.E. of three replicates of two independent experiments of normalized caspase activities of drug-treated groups to the DMSO-treated (control) groups. Statistical analyses were performed using the Student’s two tailed paired t -test and significance was determined at * p ≤ 0.05, ** p ≤ 0.01, and *** p ≤ 0.001. ( B ) Levels of expressed cleaved and total caspase-3 proteins were detected by WB analysis. Actin was used as a loading control. Dox, AD 312, and AD 198 treatments increased cleavage of the caspase-3 in wt-p53 RT4 and SW780 cells. AD 198 increased cleavage of caspase-3 in mt-p53 5637 cells, and Dox increased cleavage of caspase-3 in mt-p53 UM-UC-3 and T-24 cells. No cleaved caspase-3 was detected in mt-p53 J82 and TCCSUP cells.
    Gene Silencers generally consist of pools of three to five target specific 19 25 nucleotide sequences in length For independent verification of p53 gene silencing results individual duplex components or plasmids are also available upon request Suitable control antibody p53 Antibody A 1 sc 393031 is recommended as control antibody for monitoring of p53 expression knockdown by Western blotting or immunofluorescence
    https://www.bioz.com/result/p53/product/Santa Cruz Biotechnology
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    p53 - by Bioz Stars, 2021-06
    86/100 stars

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    1) Product Images from "Mutations of p53 decrease sensitivity to the anthracycline treatments in bladder cancer cells"

    Article Title: Mutations of p53 decrease sensitivity to the anthracycline treatments in bladder cancer cells

    Journal: Oncotarget

    doi: 10.18632/oncotarget.25530

    Dox, AD 312, and AD 198 treatments increased caspase-3/7 activity and cleaved caspase-3 protein expression in wt-p53 bladder TCC cells The wt-p53 RT4 and SW780 cells, and the mt-p53 5637, UM-UC-3, T-24, J82, and TCCSUP cells were treated with 1 µM Dox, 10 µM AD 312, and 1 µM AD 198 for 24 hours. ( A ) The activities of caspase 3/7 were measured by Caspase-Glo3/7 luminescence assay. A significant increase in caspase 3/7 activities by Dox, AD 312 and AD 198 treatments were detected in wt-p53 RT4 and SW780 cells. Only moderate upregulation of caspase 3/7 activities by Dox, AD 312 and AD 198 were observed in mt-p53 5637 cells. Dox treatment significantly increased caspase 3/7 activity in mt-p53 UM-UC-3 and T-24 cells. Low caspase 3/7 activities were detected in mt-p53 J82 and TCCSUP cells. Data shown here are mean ± S.E. of three replicates of two independent experiments of normalized caspase activities of drug-treated groups to the DMSO-treated (control) groups. Statistical analyses were performed using the Student’s two tailed paired t -test and significance was determined at * p ≤ 0.05, ** p ≤ 0.01, and *** p ≤ 0.001. ( B ) Levels of expressed cleaved and total caspase-3 proteins were detected by WB analysis. Actin was used as a loading control. Dox, AD 312, and AD 198 treatments increased cleavage of the caspase-3 in wt-p53 RT4 and SW780 cells. AD 198 increased cleavage of caspase-3 in mt-p53 5637 cells, and Dox increased cleavage of caspase-3 in mt-p53 UM-UC-3 and T-24 cells. No cleaved caspase-3 was detected in mt-p53 J82 and TCCSUP cells.
    Figure Legend Snippet: Dox, AD 312, and AD 198 treatments increased caspase-3/7 activity and cleaved caspase-3 protein expression in wt-p53 bladder TCC cells The wt-p53 RT4 and SW780 cells, and the mt-p53 5637, UM-UC-3, T-24, J82, and TCCSUP cells were treated with 1 µM Dox, 10 µM AD 312, and 1 µM AD 198 for 24 hours. ( A ) The activities of caspase 3/7 were measured by Caspase-Glo3/7 luminescence assay. A significant increase in caspase 3/7 activities by Dox, AD 312 and AD 198 treatments were detected in wt-p53 RT4 and SW780 cells. Only moderate upregulation of caspase 3/7 activities by Dox, AD 312 and AD 198 were observed in mt-p53 5637 cells. Dox treatment significantly increased caspase 3/7 activity in mt-p53 UM-UC-3 and T-24 cells. Low caspase 3/7 activities were detected in mt-p53 J82 and TCCSUP cells. Data shown here are mean ± S.E. of three replicates of two independent experiments of normalized caspase activities of drug-treated groups to the DMSO-treated (control) groups. Statistical analyses were performed using the Student’s two tailed paired t -test and significance was determined at * p ≤ 0.05, ** p ≤ 0.01, and *** p ≤ 0.001. ( B ) Levels of expressed cleaved and total caspase-3 proteins were detected by WB analysis. Actin was used as a loading control. Dox, AD 312, and AD 198 treatments increased cleavage of the caspase-3 in wt-p53 RT4 and SW780 cells. AD 198 increased cleavage of caspase-3 in mt-p53 5637 cells, and Dox increased cleavage of caspase-3 in mt-p53 UM-UC-3 and T-24 cells. No cleaved caspase-3 was detected in mt-p53 J82 and TCCSUP cells.

    Techniques Used: Activity Assay, Expressing, Luminescence Assay, Two Tailed Test, Western Blot

    Dox, AD 312, and AD 198 treatments increased p53 protein expression in wt-p53 bladder TCC cells Human bladder TCC cells were treated with 0.1 µM, 0.5 µM, and 1 µM of Dox or AD 198 or 1 µM, 5 µM, and 10 µM of AD 312 in serum free media for 24 hours and levels of p53 protein expression were detected by WB analysis. Actin was used as a loading control. ( A ) Significant increase in p53 protein levels were detected in wt-p53 RT4 and SW780 cells after anthracycline treatments in a dose-dependent manner. ( B ) Slight to no change in the expression of p53 protein levels were detected in the mt-p53 5637, UM-UC-3, T-24, J82, and TCCSUP cells after either anthracycline treatment. Densitometry analysis of normalized p53 protein levels to actin and relative to the DMSO-treated (control) groups are shown as means ± S.E. of three readings of two independent experiments. Statistical analyses were performed using the Student’s two tailed paired t -test and significance was determined at * p ≤ 0.05, ** p ≤ 0.01, and *** p ≤ 0.001.
    Figure Legend Snippet: Dox, AD 312, and AD 198 treatments increased p53 protein expression in wt-p53 bladder TCC cells Human bladder TCC cells were treated with 0.1 µM, 0.5 µM, and 1 µM of Dox or AD 198 or 1 µM, 5 µM, and 10 µM of AD 312 in serum free media for 24 hours and levels of p53 protein expression were detected by WB analysis. Actin was used as a loading control. ( A ) Significant increase in p53 protein levels were detected in wt-p53 RT4 and SW780 cells after anthracycline treatments in a dose-dependent manner. ( B ) Slight to no change in the expression of p53 protein levels were detected in the mt-p53 5637, UM-UC-3, T-24, J82, and TCCSUP cells after either anthracycline treatment. Densitometry analysis of normalized p53 protein levels to actin and relative to the DMSO-treated (control) groups are shown as means ± S.E. of three readings of two independent experiments. Statistical analyses were performed using the Student’s two tailed paired t -test and significance was determined at * p ≤ 0.05, ** p ≤ 0.01, and *** p ≤ 0.001.

    Techniques Used: Expressing, Western Blot, Two Tailed Test

    AD 198 decreased c-myc protein levels in mt-p53 bladder TCC cells Human bladder TCC cells were treated with 0.1 µM, 0.5 µM, and 1 µM of Dox or AD 198 or 1 µM, 5 µM, and 10 µM of AD 312 in serum free media for 24 hours and levels of c-myc protein expression were detected by WB analysis. Actin was used as a loading control. ( A ) No levels of c-myc protein expression were detected in the wt-p53 RT4 and SW780 cells after anthracycline treatments. UM-UC-3 cells (lane 1) expressing c-myc protein were used as a positive control. ( B ) Significant decrease of c-myc protein expression was detected in the mt-p53 5637, UM-UC-3, T-24, and J82 cells after AD 198 treatment in a dose-dependent manner. Densitometry analysis of normalized c-myc protein levels to actin followed by normalization of anthracyclines treated to control groups are shown in chart as means ± S.E. of three readings of two independent experiments. Statistical analyses were performed using the Student’s two tailed paired t -test and significance was determined at * p ≤ 0.05, ** p ≤ 0.01, and *** p ≤ 0.001.
    Figure Legend Snippet: AD 198 decreased c-myc protein levels in mt-p53 bladder TCC cells Human bladder TCC cells were treated with 0.1 µM, 0.5 µM, and 1 µM of Dox or AD 198 or 1 µM, 5 µM, and 10 µM of AD 312 in serum free media for 24 hours and levels of c-myc protein expression were detected by WB analysis. Actin was used as a loading control. ( A ) No levels of c-myc protein expression were detected in the wt-p53 RT4 and SW780 cells after anthracycline treatments. UM-UC-3 cells (lane 1) expressing c-myc protein were used as a positive control. ( B ) Significant decrease of c-myc protein expression was detected in the mt-p53 5637, UM-UC-3, T-24, and J82 cells after AD 198 treatment in a dose-dependent manner. Densitometry analysis of normalized c-myc protein levels to actin followed by normalization of anthracyclines treated to control groups are shown in chart as means ± S.E. of three readings of two independent experiments. Statistical analyses were performed using the Student’s two tailed paired t -test and significance was determined at * p ≤ 0.05, ** p ≤ 0.01, and *** p ≤ 0.001.

    Techniques Used: Expressing, Western Blot, Positive Control, Two Tailed Test

    p53 siRNA blocked Dox-, AD 312-, and AD 198-induced cPARP in wt-p53 RT4 cells The wt-p53 bladder TCC RT4 cells were transfected with 75 nM p53 siRNA and control siRNA. Twenty-four hours after transfection, the cells were treated with DMSO (control), Dox (1 µM), AD 312 (10 µM), or AD 198 (1 µM) in serum free media for an additional 24 hours. The expression levels of p53 and cPARP were detected by WB analysis. Actin was used as a loading control. p53 siRNA transfection blocked Dox-, AD 312-, and AD 198-induced cPARP in wt-p53 bladder TCC RT4 cells. Densitometry analysis of normalized p53 or cPARP protein levels to actin and relative to the control-treated (DMSO) groups are shown as means ± S.E. of three readings of three independent experiments. Statistical analyses were performed using the Student’s two tailed paired t -test and significance was determined at * p ≤ 0.05, ** p ≤ 0.01 and *** p ≤ 0.001.
    Figure Legend Snippet: p53 siRNA blocked Dox-, AD 312-, and AD 198-induced cPARP in wt-p53 RT4 cells The wt-p53 bladder TCC RT4 cells were transfected with 75 nM p53 siRNA and control siRNA. Twenty-four hours after transfection, the cells were treated with DMSO (control), Dox (1 µM), AD 312 (10 µM), or AD 198 (1 µM) in serum free media for an additional 24 hours. The expression levels of p53 and cPARP were detected by WB analysis. Actin was used as a loading control. p53 siRNA transfection blocked Dox-, AD 312-, and AD 198-induced cPARP in wt-p53 bladder TCC RT4 cells. Densitometry analysis of normalized p53 or cPARP protein levels to actin and relative to the control-treated (DMSO) groups are shown as means ± S.E. of three readings of three independent experiments. Statistical analyses were performed using the Student’s two tailed paired t -test and significance was determined at * p ≤ 0.05, ** p ≤ 0.01 and *** p ≤ 0.001.

    Techniques Used: Transfection, Expressing, Western Blot, Two Tailed Test

    Dox, AD 312, and AD 198 treatments increased cleaved PARP (cPARP) in wt-p53 bladder TCC cells ( A ) The wt-p53 and ( B ) mt-p53 bladder TCC cells were treated with 0.1 µM, 0.5 µM, and 1 µM of Dox or AD 198 or 1 µM, 5 µM, and 10 µM of AD 312 in serum free media for 24 hours and cPARP expression was detected by WB analysis. Actin was used as a loading control. The increased cPARP expression was detected after high doses of all drug treatments in RT4 cells and after high dose of AD 198 in SW780 cells. Antracycline treatments did not change the levels of cPARP protein in 5637 cells. Dox treatment upregulated cPARP in UM-UC-3 and T-24 cells and no cPARP was detected in J82 and TCCSUP cells. Densitometry analysis of normalized cPARP protein levels to actin and relative to the DMSO-treated (control) groups are shown as means ± S.E. of three readings of two independent experiments. Statistical analyses were performed using the Student’s two tailed paired t -test and significance was determined at * p ≤ 0.05 and ** p ≤ 0.01.
    Figure Legend Snippet: Dox, AD 312, and AD 198 treatments increased cleaved PARP (cPARP) in wt-p53 bladder TCC cells ( A ) The wt-p53 and ( B ) mt-p53 bladder TCC cells were treated with 0.1 µM, 0.5 µM, and 1 µM of Dox or AD 198 or 1 µM, 5 µM, and 10 µM of AD 312 in serum free media for 24 hours and cPARP expression was detected by WB analysis. Actin was used as a loading control. The increased cPARP expression was detected after high doses of all drug treatments in RT4 cells and after high dose of AD 198 in SW780 cells. Antracycline treatments did not change the levels of cPARP protein in 5637 cells. Dox treatment upregulated cPARP in UM-UC-3 and T-24 cells and no cPARP was detected in J82 and TCCSUP cells. Densitometry analysis of normalized cPARP protein levels to actin and relative to the DMSO-treated (control) groups are shown as means ± S.E. of three readings of two independent experiments. Statistical analyses were performed using the Student’s two tailed paired t -test and significance was determined at * p ≤ 0.05 and ** p ≤ 0.01.

    Techniques Used: Expressing, Western Blot, Two Tailed Test

    PRIMA-1 sensitized mt-p53 J82, but not wt-p53 RT4 cells to AD 198 treatment ( A ) wt-p53 RT4 and mt-p53 J82 cells were treated with increasing doses of PRIMA-1 (5, 10, 15, 20, and 25 µM) for 48 hours and cell viability was assessed by MTS assay. PRIMA-1 treatments did not affect the cell viability of wt-p53 RT4 cells. On the other hand, PRIMA-1 treatment decreased cell viability in a dose-dependent manner in mt-p53 J82 cells. ( B ) PRIMA-1 treatment (10 µM) upregulated p21 protein levels in mt-p53 J82 cells, but not in wt-p53 RT4 cells. ( C ) RT4 and J82 cells were treated with 10 µM PRIMA-1 alone or in combination with 0.5 µM of Dox (left panel) or AD 198 (right panel) for 48 hours and cell viability was assessed by MTS assay. A co-treatment of AD 198 with PRIMA-1 highly significantly decreased cell viability as compared to AD 198 treatment alone in mt-p53 J82 cells ( ### p ≤ 0.001). Values shown as means ± S.E. of four replicates of two independent experiments of normalized cell viability of treated groups to control (DMSO) group. ( D ) The activity of caspase 3/7 in mt-p53 J82 cells were measured by Caspase-Glo3/7 luminescence assay after treatments. Co-treatment of AD 198 with PRIMA-1 increased activities of caspase-3/7. Data shown here are normalized caspase activities of drug-treated groups to control groups as means ± S.E. of three replicates of two independent experiments. Statistical analyses were performed using the Student’s two tailed paired t -test and significance was determined comparing treatment to control groups ( * p
    Figure Legend Snippet: PRIMA-1 sensitized mt-p53 J82, but not wt-p53 RT4 cells to AD 198 treatment ( A ) wt-p53 RT4 and mt-p53 J82 cells were treated with increasing doses of PRIMA-1 (5, 10, 15, 20, and 25 µM) for 48 hours and cell viability was assessed by MTS assay. PRIMA-1 treatments did not affect the cell viability of wt-p53 RT4 cells. On the other hand, PRIMA-1 treatment decreased cell viability in a dose-dependent manner in mt-p53 J82 cells. ( B ) PRIMA-1 treatment (10 µM) upregulated p21 protein levels in mt-p53 J82 cells, but not in wt-p53 RT4 cells. ( C ) RT4 and J82 cells were treated with 10 µM PRIMA-1 alone or in combination with 0.5 µM of Dox (left panel) or AD 198 (right panel) for 48 hours and cell viability was assessed by MTS assay. A co-treatment of AD 198 with PRIMA-1 highly significantly decreased cell viability as compared to AD 198 treatment alone in mt-p53 J82 cells ( ### p ≤ 0.001). Values shown as means ± S.E. of four replicates of two independent experiments of normalized cell viability of treated groups to control (DMSO) group. ( D ) The activity of caspase 3/7 in mt-p53 J82 cells were measured by Caspase-Glo3/7 luminescence assay after treatments. Co-treatment of AD 198 with PRIMA-1 increased activities of caspase-3/7. Data shown here are normalized caspase activities of drug-treated groups to control groups as means ± S.E. of three replicates of two independent experiments. Statistical analyses were performed using the Student’s two tailed paired t -test and significance was determined comparing treatment to control groups ( * p

    Techniques Used: MTS Assay, Activity Assay, Luminescence Assay, Two Tailed Test

    2) Product Images from "Increasing cisplatin sensitivity by schedule-dependent inhibition of AKT and Chk1"

    Article Title: Increasing cisplatin sensitivity by schedule-dependent inhibition of AKT and Chk1

    Journal: Cancer Biology & Therapy

    doi: 10.4161/15384047.2014.961876

    Chk1 inhibitor sensitizes OS cells to cisplatin. ( A ) OS cells lines were treated with CP (5 μM for SJSA1 and MG63, 10 μM for MHM and U2OS, 2 μM for SAOS) for 48 hours. Whole cell lysates were immunoblotted for pChk1 (S296 and S345), Chk1, p53, and β-actin. ( B ) The cells were treated with CP (5 μM for SJSA1 and MG63, 10 μM for MHM, 15 μM for U2OS, 2 μM for SAOS) and LY2603618 (LY, 0.2 μM) singly or combined for 72 hours and analyzed with FACS for sub-G1 apoptosis. Average percent apoptotic cells from triplicate were presented as a graph with standard deviation indicated. There is significant difference between CP and CP plus LY ( p
    Figure Legend Snippet: Chk1 inhibitor sensitizes OS cells to cisplatin. ( A ) OS cells lines were treated with CP (5 μM for SJSA1 and MG63, 10 μM for MHM and U2OS, 2 μM for SAOS) for 48 hours. Whole cell lysates were immunoblotted for pChk1 (S296 and S345), Chk1, p53, and β-actin. ( B ) The cells were treated with CP (5 μM for SJSA1 and MG63, 10 μM for MHM, 15 μM for U2OS, 2 μM for SAOS) and LY2603618 (LY, 0.2 μM) singly or combined for 72 hours and analyzed with FACS for sub-G1 apoptosis. Average percent apoptotic cells from triplicate were presented as a graph with standard deviation indicated. There is significant difference between CP and CP plus LY ( p

    Techniques Used: FACS, Standard Deviation

    P53 knockdown reduces Noxa and Puma gene expression in cells treated with CP or CP plus MK2206. ( A ) MHM cells were transiently transfected with control siRNA (Csi) or p53 siRNA (p53si) were treated with CP (10 μM) or CP plus MK for 48 hours. Relative Noxa and Puma gene expression was analyzed by qTPCR and presented as graphs. ( B ) Whole cell lysates were immunoblotted for p53 and β-actin. ( C ) U2OS cells expressing control shRNA (Csh) or p53 shRNA (p53sh) were treated with CP (15 μM) or CP plus MK for 48 hours. Relative Noxa and Puma genes were analyzed by qTPCR and presented as graphs. ( D ) Whole cell lysates were immunoblotted for p53 and β-actin.
    Figure Legend Snippet: P53 knockdown reduces Noxa and Puma gene expression in cells treated with CP or CP plus MK2206. ( A ) MHM cells were transiently transfected with control siRNA (Csi) or p53 siRNA (p53si) were treated with CP (10 μM) or CP plus MK for 48 hours. Relative Noxa and Puma gene expression was analyzed by qTPCR and presented as graphs. ( B ) Whole cell lysates were immunoblotted for p53 and β-actin. ( C ) U2OS cells expressing control shRNA (Csh) or p53 shRNA (p53sh) were treated with CP (15 μM) or CP plus MK for 48 hours. Relative Noxa and Puma genes were analyzed by qTPCR and presented as graphs. ( D ) Whole cell lysates were immunoblotted for p53 and β-actin.

    Techniques Used: Expressing, Transfection, shRNA, Cell Surface Hydrophobicity

    CP induces loss of p27 which is prevented by MK2206. Knockdown of p27 reduces MK2206-mediated G1 arrest and sensitized p53-null cells to apoptosis in response to CP plus MK. ( A ) MG63, SAOS, U2OS cells were treated with CP and/or MK2206 for 24 hours. Whole cell lysates were immunoblotted for p27 and β-actin. ( B ) MG63 cells were transiently transfected with control siRNA (Csi) or p27kip siRNA (p27si) and treated with CP (5 μM) and/or MK2206 for 72 hours. The cells were stained by PI and analyzed for cell cycle. Representative cell cycle profiles are shown. The table below the cell cycle profiles shows the average percent G1, S, and G2/M populations from triplicate experiments +/− s.e.m. ( C ) Upper Percent sub-G1 cell (+/− s.e.m.) is shown for control and p27 knockdown MG63 cells treated with CP, MK, or CP plus MK for 72 hrs. There is significant difference in Sub-G1 and G1 population between Csi and p27si ( p
    Figure Legend Snippet: CP induces loss of p27 which is prevented by MK2206. Knockdown of p27 reduces MK2206-mediated G1 arrest and sensitized p53-null cells to apoptosis in response to CP plus MK. ( A ) MG63, SAOS, U2OS cells were treated with CP and/or MK2206 for 24 hours. Whole cell lysates were immunoblotted for p27 and β-actin. ( B ) MG63 cells were transiently transfected with control siRNA (Csi) or p27kip siRNA (p27si) and treated with CP (5 μM) and/or MK2206 for 72 hours. The cells were stained by PI and analyzed for cell cycle. Representative cell cycle profiles are shown. The table below the cell cycle profiles shows the average percent G1, S, and G2/M populations from triplicate experiments +/− s.e.m. ( C ) Upper Percent sub-G1 cell (+/− s.e.m.) is shown for control and p27 knockdown MG63 cells treated with CP, MK, or CP plus MK for 72 hrs. There is significant difference in Sub-G1 and G1 population between Csi and p27si ( p

    Techniques Used: Transfection, Staining

    Cisplatin (CP) activates AKT and Chk1 in OS cells. ( A ) OS cells lines were treated with CP (5 μM for SJSA1 and MG63, 10 μM for MHM and U2OS, 2 μM for SAOS) for 48 hours. Whole cell lysates were immunoblotted for pAKT (S473), total AKT, pChk1 (S345), Chk1, p53, and β-actin. ( B ) The cells were treated with indicated doses of CP for 72 hours and analyzed with FACS for sub-G1 apoptosis. Average percent apoptotic cells from triplicate were presented as a graph with standard deviation indicated. (Representative of at least 3 independent experiments).
    Figure Legend Snippet: Cisplatin (CP) activates AKT and Chk1 in OS cells. ( A ) OS cells lines were treated with CP (5 μM for SJSA1 and MG63, 10 μM for MHM and U2OS, 2 μM for SAOS) for 48 hours. Whole cell lysates were immunoblotted for pAKT (S473), total AKT, pChk1 (S345), Chk1, p53, and β-actin. ( B ) The cells were treated with indicated doses of CP for 72 hours and analyzed with FACS for sub-G1 apoptosis. Average percent apoptotic cells from triplicate were presented as a graph with standard deviation indicated. (Representative of at least 3 independent experiments).

    Techniques Used: FACS, Standard Deviation

    Inhibition of AKT increases CP-induced apoptosis in OS cells expressing WT p53 but not p53-null cells. ( A ) OS cells lines were treated with CP alone (5 μM for SJSA1 and MG63, 10 μM for MHM and U2OS, 2 μM for SAOS) or CP plus MK2206 (MK, 10 μM) for 48 hours. Whole cell lysates were immunoblotted for pAKT (S473), p53, and β-actin. ( B ) The cells were treated with CP and MK2206 (doses as above) singly or combined for 72 hours and analyzed with FACS for sub-G1 apoptosis. Average percent apoptotic cells from triplicate experiments is graphed with standard deviation indicated. There is significant difference between CP and CP plus MK ( p
    Figure Legend Snippet: Inhibition of AKT increases CP-induced apoptosis in OS cells expressing WT p53 but not p53-null cells. ( A ) OS cells lines were treated with CP alone (5 μM for SJSA1 and MG63, 10 μM for MHM and U2OS, 2 μM for SAOS) or CP plus MK2206 (MK, 10 μM) for 48 hours. Whole cell lysates were immunoblotted for pAKT (S473), p53, and β-actin. ( B ) The cells were treated with CP and MK2206 (doses as above) singly or combined for 72 hours and analyzed with FACS for sub-G1 apoptosis. Average percent apoptotic cells from triplicate experiments is graphed with standard deviation indicated. There is significant difference between CP and CP plus MK ( p

    Techniques Used: Inhibition, Expressing, FACS, Standard Deviation

    Scheduled inhibition of AKT reduces p27, abrogates G1 arrest, and sensitizes p53-null cells to CP-induced apoptosis. ( A ) Cells were treated with CP (5 μM for MG63, 2 μM for SAOS) alone, CP plus MK2206 simultaneously (SIM), or CP first for 24 hrs followed by MK2206 (sequentially; SEQ) for 48 hrs. Whole cells lysates were immunoblotted for p27, pAKT, total AKT, and β-actin. ( B and C ) MG63 and SAOS cells were treated as indicated for 72 hours, and cell cycle determined by FACS. Representative histograms are shown. The tables below the histograms shows the average percent sub-G1, G1, S, and G2/M populations from triplicate experiments +/− s.e.m. There is significant difference in sub-G1 and G1 population ( p
    Figure Legend Snippet: Scheduled inhibition of AKT reduces p27, abrogates G1 arrest, and sensitizes p53-null cells to CP-induced apoptosis. ( A ) Cells were treated with CP (5 μM for MG63, 2 μM for SAOS) alone, CP plus MK2206 simultaneously (SIM), or CP first for 24 hrs followed by MK2206 (sequentially; SEQ) for 48 hrs. Whole cells lysates were immunoblotted for p27, pAKT, total AKT, and β-actin. ( B and C ) MG63 and SAOS cells were treated as indicated for 72 hours, and cell cycle determined by FACS. Representative histograms are shown. The tables below the histograms shows the average percent sub-G1, G1, S, and G2/M populations from triplicate experiments +/− s.e.m. There is significant difference in sub-G1 and G1 population ( p

    Techniques Used: Inhibition, FACS

    3) Product Images from "Cadmium Modifies the Cell Cycle and Apoptotic Profiles of Human Breast Cancer Cells Treated with 5-Fluorouracil"

    Article Title: Cadmium Modifies the Cell Cycle and Apoptotic Profiles of Human Breast Cancer Cells Treated with 5-Fluorouracil

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms140816600

    Determination by qRT-PCR of gene expression after treatment with Cd and/or 5-FU for 6, 24, or 48 h. The fold-increase in expression was quantified after normalizing expression levels for those in control MCF-7 cells. Gene expression of bcl-2 ( A ); p53 ( B ); bax ( C ); caspase 8 ( D ); caspase 9 ( E ); c-myc ( F ); cyclin D1 ( G ); cyclin A1 ( H ).
    Figure Legend Snippet: Determination by qRT-PCR of gene expression after treatment with Cd and/or 5-FU for 6, 24, or 48 h. The fold-increase in expression was quantified after normalizing expression levels for those in control MCF-7 cells. Gene expression of bcl-2 ( A ); p53 ( B ); bax ( C ); caspase 8 ( D ); caspase 9 ( E ); c-myc ( F ); cyclin D1 ( G ); cyclin A1 ( H ).

    Techniques Used: Quantitative RT-PCR, Expressing

    4) Product Images from "Evidence for Phenotypic Plasticity in Aggressive Triple-Negative Breast Cancer: Human Biology Is Recapitulated by a Novel Model System"

    Article Title: Evidence for Phenotypic Plasticity in Aggressive Triple-Negative Breast Cancer: Human Biology Is Recapitulated by a Novel Model System

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0045684

    Basal/myoepithelial differentiation of DKAT xenografts. A. DKAT xenograft tumor stained with DAPI (blue) nuclear counter stain, CK14 (green) showing basal/myoepithelial differentiation of tumor cells, and human-specific p53 (red) confirming that both the CK14-positive cells and the more luminal, CK14 weak/negative cells are human DKAT tumor cells. 100 micron scale bar in the DAPI channel applies to all four images. B. DKAT tumor from a second mouse at higher magnification shows an area of vague glandular architecture with somewhat elongated CK14-positive basal/myoepithelial cells (arrows) surrounding CK14 weak/negative cells which surround a poorly formed glandular lumen (asterix). 25 micron scale bar show in in DAPI channel.
    Figure Legend Snippet: Basal/myoepithelial differentiation of DKAT xenografts. A. DKAT xenograft tumor stained with DAPI (blue) nuclear counter stain, CK14 (green) showing basal/myoepithelial differentiation of tumor cells, and human-specific p53 (red) confirming that both the CK14-positive cells and the more luminal, CK14 weak/negative cells are human DKAT tumor cells. 100 micron scale bar in the DAPI channel applies to all four images. B. DKAT tumor from a second mouse at higher magnification shows an area of vague glandular architecture with somewhat elongated CK14-positive basal/myoepithelial cells (arrows) surrounding CK14 weak/negative cells which surround a poorly formed glandular lumen (asterix). 25 micron scale bar show in in DAPI channel.

    Techniques Used: Staining

    5) Product Images from "Prep1 Directly Regulates the Intrinsic Apoptotic Pathway by Controlling Bcl-XL Levels ▿ Levels ▿ †"

    Article Title: Prep1 Directly Regulates the Intrinsic Apoptotic Pathway by Controlling Bcl-XL Levels ▿ Levels ▿ †

    Journal:

    doi: 10.1128/MCB.01273-08

    The p53-dependent response to genotoxic stress is slightly affected in Prep1 i/i MEFs. (A) Total RNA extracted from untreated MEFs was obtained from Prep1 +/+ (+/+) ( n = 4) and Prep1 i/i (i/i) ( n = 4) embryos
    Figure Legend Snippet: The p53-dependent response to genotoxic stress is slightly affected in Prep1 i/i MEFs. (A) Total RNA extracted from untreated MEFs was obtained from Prep1 +/+ (+/+) ( n = 4) and Prep1 i/i (i/i) ( n = 4) embryos

    Techniques Used:

    6) Product Images from "Inhibitory Effect of Lycopene on Amyloid-β-Induced Apoptosis in Neuronal Cells"

    Article Title: Inhibitory Effect of Lycopene on Amyloid-β-Induced Apoptosis in Neuronal Cells

    Journal: Nutrients

    doi: 10.3390/nu9080883

    Effect of lycopene on cell viability and apoptotic indices in amyloid-β-stimulated cells. The cells were pretreated with lycopene for 1 h and then stimulated with amyloid-β (20 μM) for another 24 h. ( A ) Viable cell numbers was determined by the Trypan Blue exclusion test; ( B ) Levels of p53, Bax, Bcl-2, caspase-3, and actin were determined by western blot analysis; ( C ) The ratio of Bax/Bcl-2 was determined by protein band densities of Bax and Bcl-2; ( D ) Levels of pro- and cleaved-caspase-3 were assessed by western blot analysis. Actin was used as a loading control. The value of None (without any stimulation or treatment) was set as 100%. Data are expressed as the mean ± S.E. of three independent experiments. ** p
    Figure Legend Snippet: Effect of lycopene on cell viability and apoptotic indices in amyloid-β-stimulated cells. The cells were pretreated with lycopene for 1 h and then stimulated with amyloid-β (20 μM) for another 24 h. ( A ) Viable cell numbers was determined by the Trypan Blue exclusion test; ( B ) Levels of p53, Bax, Bcl-2, caspase-3, and actin were determined by western blot analysis; ( C ) The ratio of Bax/Bcl-2 was determined by protein band densities of Bax and Bcl-2; ( D ) Levels of pro- and cleaved-caspase-3 were assessed by western blot analysis. Actin was used as a loading control. The value of None (without any stimulation or treatment) was set as 100%. Data are expressed as the mean ± S.E. of three independent experiments. ** p

    Techniques Used: Western Blot

    Cell viability and apoptotic indices in NT siRNA-transfected cells and Nucling siRNA-transfected cells with or without amyloid-β stimulation. The transfected cells were pre-treated with lycopene for 1 h and, then, stimulated with amyloid-β (20 μM) for another 24 h. ( A ) Levels of Nucling were determined by western blot analysis. Protein levels of Nucling were compared with that of the loading control actin and expressed as percentage ratios of the band densities; ( B ) Viable cell number was determined by the Trypan Blue exclusion test. The value for NT siRNA-transfected cells without amyloid-β stimulation was set as 100%; ( C ) Levels of p53, Bax and Bcl-2 were determined by western blot analysis. The ratio of Bax/Bcl-2 was determined by protein band densities of Bax and Bcl-2; ( D ) The levels of pro- and cleaved-caspase-3 were determined by western blot analysis. Actin served as a loading control. Values are expressed as the mean ± S.E. of three independent experiments. ** p
    Figure Legend Snippet: Cell viability and apoptotic indices in NT siRNA-transfected cells and Nucling siRNA-transfected cells with or without amyloid-β stimulation. The transfected cells were pre-treated with lycopene for 1 h and, then, stimulated with amyloid-β (20 μM) for another 24 h. ( A ) Levels of Nucling were determined by western blot analysis. Protein levels of Nucling were compared with that of the loading control actin and expressed as percentage ratios of the band densities; ( B ) Viable cell number was determined by the Trypan Blue exclusion test. The value for NT siRNA-transfected cells without amyloid-β stimulation was set as 100%; ( C ) Levels of p53, Bax and Bcl-2 were determined by western blot analysis. The ratio of Bax/Bcl-2 was determined by protein band densities of Bax and Bcl-2; ( D ) The levels of pro- and cleaved-caspase-3 were determined by western blot analysis. Actin served as a loading control. Values are expressed as the mean ± S.E. of three independent experiments. ** p

    Techniques Used: Transfection, Western Blot

    7) Product Images from "Treatment with Caffeic Acid and Resveratrol Alleviates Oxidative Stress Induced Neurotoxicity in Cell and Drosophila Models of Spinocerebellar Ataxia Type3"

    Article Title: Treatment with Caffeic Acid and Resveratrol Alleviates Oxidative Stress Induced Neurotoxicity in Cell and Drosophila Models of Spinocerebellar Ataxia Type3

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-11839-0

    Effects of CA and Res in tBH-treated SK-N-SH-MJD78 cells transfected with dominant-negative mutant IκB-α. Cells were transiently transfected with IκB-α wild-type (WT) or DNM IκB-α as well as with or without reporter genes of p53-Luc or pNF-κB-Luc for 16 h and were then treated with either vehicle control or tBH plus CA or Res. (A) Phosphorylated and total p53 and IκB-α as well as nuclear p53 and p65 (3-h and 4-h treatments for p53 and NF-κB activation, respectively). (B) Protein expression of mutant and normal ataxin-3 and Hsp27. (C) Protein expression of Bax, Bcl-2, and pro and cleaved caspase 3 and PARP. (D) Levels of NF-κB and p53 reporter gene activities, protein aggregates, cell apoptosis rates, and caspase 3 activity. Data are the mean ± SD. Within treatments with the same plasmid transfection, data are expressed as the percentage of the SK-N-SH-MJD78 cells treated with tBH alone, and values not having the same letter are significantly different (p
    Figure Legend Snippet: Effects of CA and Res in tBH-treated SK-N-SH-MJD78 cells transfected with dominant-negative mutant IκB-α. Cells were transiently transfected with IκB-α wild-type (WT) or DNM IκB-α as well as with or without reporter genes of p53-Luc or pNF-κB-Luc for 16 h and were then treated with either vehicle control or tBH plus CA or Res. (A) Phosphorylated and total p53 and IκB-α as well as nuclear p53 and p65 (3-h and 4-h treatments for p53 and NF-κB activation, respectively). (B) Protein expression of mutant and normal ataxin-3 and Hsp27. (C) Protein expression of Bax, Bcl-2, and pro and cleaved caspase 3 and PARP. (D) Levels of NF-κB and p53 reporter gene activities, protein aggregates, cell apoptosis rates, and caspase 3 activity. Data are the mean ± SD. Within treatments with the same plasmid transfection, data are expressed as the percentage of the SK-N-SH-MJD78 cells treated with tBH alone, and values not having the same letter are significantly different (p

    Techniques Used: Transfection, Dominant Negative Mutation, Activation Assay, Expressing, Mutagenesis, Activity Assay, Plasmid Preparation

    Effects of CA and Res on activation of p53 and NF-κB in tBH-treated SK-N-SH-MJD78 cells. (A) and (B) Protein expression of phosphorylated and total p53, IKK-β, IκB-α, and nuclear p53 and p65. (C) NF-κB and p53 reporter gene activities (3-h and 4-h treatments for p53 and NF-κB activation, respectively). Data are the mean ± SD and are expressed as the percentage of SK-N-SH-MJD78 cells treated with tBH alone. Values not having the same letter are significantly different (p
    Figure Legend Snippet: Effects of CA and Res on activation of p53 and NF-κB in tBH-treated SK-N-SH-MJD78 cells. (A) and (B) Protein expression of phosphorylated and total p53, IKK-β, IκB-α, and nuclear p53 and p65. (C) NF-κB and p53 reporter gene activities (3-h and 4-h treatments for p53 and NF-κB activation, respectively). Data are the mean ± SD and are expressed as the percentage of SK-N-SH-MJD78 cells treated with tBH alone. Values not having the same letter are significantly different (p

    Techniques Used: Activation Assay, Expressing

    Effects of CA and Res on tBH-treated ELAV-SCA3tr-Q78 transgenic Drosophila . (A) H 2 DCFDA, MitoSOX, and protein aggregate levels. (B) Mutant and normal ataxin-3, Hsp27, Bax, Bcl-2. (C) p53 and NF-κB protein expression in 22-day-old male ELAV-SCA3tr-Q78 flies. Values are mean ± SD, n = 50 male flies in three separate experiments. Data are expressed as the percentage of ELAV-SCA3tr-Q78 flies treated with tBH alone. Values not having the same letter are significantly different (p
    Figure Legend Snippet: Effects of CA and Res on tBH-treated ELAV-SCA3tr-Q78 transgenic Drosophila . (A) H 2 DCFDA, MitoSOX, and protein aggregate levels. (B) Mutant and normal ataxin-3, Hsp27, Bax, Bcl-2. (C) p53 and NF-κB protein expression in 22-day-old male ELAV-SCA3tr-Q78 flies. Values are mean ± SD, n = 50 male flies in three separate experiments. Data are expressed as the percentage of ELAV-SCA3tr-Q78 flies treated with tBH alone. Values not having the same letter are significantly different (p

    Techniques Used: Transgenic Assay, Mutagenesis, Expressing

    8) Product Images from "Targeted Inhibition of Multiple Receptor Tyrosine Kinases in Mesothelioma 1Targeted Inhibition of Multiple Receptor Tyrosine Kinases in Mesothelioma 1 2"

    Article Title: Targeted Inhibition of Multiple Receptor Tyrosine Kinases in Mesothelioma 1Targeted Inhibition of Multiple Receptor Tyrosine Kinases in Mesothelioma 1 2

    Journal: Neoplasia (New York, N.Y.)

    doi:

    Effects of HSP90 inhibition (17-AAG) in mesothelioma cell lines by 17-AAG treatment for 48 hours in serum-containing medium. (A) EGFR, AXL, and MET expression. (B) p27, p53, and p21 expression and cleavage of apoptosis biomarker caspase 3. β-Actin is a loading control.
    Figure Legend Snippet: Effects of HSP90 inhibition (17-AAG) in mesothelioma cell lines by 17-AAG treatment for 48 hours in serum-containing medium. (A) EGFR, AXL, and MET expression. (B) p27, p53, and p21 expression and cleavage of apoptosis biomarker caspase 3. β-Actin is a loading control.

    Techniques Used: Inhibition, Expressing, Biomarker Assay

    9) Product Images from "Regulation of lymphocyte progenitor survival by the proapoptotic activities of Bim and Bid"

    Article Title: Regulation of lymphocyte progenitor survival by the proapoptotic activities of Bim and Bid

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.0807557106

    Direct regulation of Bid expression by p53. ( A ) Quantitative RT-PCRs for Bid and p53 expression; p53 protein expression is also shown ( Right , Upper ). *, P
    Figure Legend Snippet: Direct regulation of Bid expression by p53. ( A ) Quantitative RT-PCRs for Bid and p53 expression; p53 protein expression is also shown ( Right , Upper ). *, P

    Techniques Used: Expressing

    10) Product Images from "Integrated high-throughput analysis identifies Sp1 as a crucial determinant of p53-mediated apoptosis"

    Article Title: Integrated high-throughput analysis identifies Sp1 as a crucial determinant of p53-mediated apoptosis

    Journal: Cell Death and Differentiation

    doi: 10.1038/cdd.2014.69

    p53-mediated pro-apoptotic transcriptional repression requires the co-binding of Sp1 to p53 target genes. ( a ) Heatmap representation of the 1078 genes whose repression by RITA was lost in Sp1-depleted cells. ( b ) Proportion of p53-bound genes (p53 targets) among these 1078 genes is illustrated via Venn diagram. ( c ) Heatmap shows p53 occupancy within ±500 bp of the putative Sp1 RE in 227 p53-bound genes. ‘0' indicates the position of the putative Sp1 RE, rows indicate p53 binding (red: high peaks/binding; white: no peaks/binding). ( d ) Synthetic lethal pathways enriched with 227 p53-bound genes after RITA treatment. P
    Figure Legend Snippet: p53-mediated pro-apoptotic transcriptional repression requires the co-binding of Sp1 to p53 target genes. ( a ) Heatmap representation of the 1078 genes whose repression by RITA was lost in Sp1-depleted cells. ( b ) Proportion of p53-bound genes (p53 targets) among these 1078 genes is illustrated via Venn diagram. ( c ) Heatmap shows p53 occupancy within ±500 bp of the putative Sp1 RE in 227 p53-bound genes. ‘0' indicates the position of the putative Sp1 RE, rows indicate p53 binding (red: high peaks/binding; white: no peaks/binding). ( d ) Synthetic lethal pathways enriched with 227 p53-bound genes after RITA treatment. P

    Techniques Used: Binding Assay

    Nutlin decreases Sp1 protein stability in p53-independent manner. ( a ) Protein levels of MDM2, Sp1 and p53 in MCF-7, HCT116 and HCT116 TP53−/− cells with RITA or nutlin treatment were assessed by immunoblotting. ( b ) mRNA level of Sp1 in MCF-7 cells with or without Sp1 depletion on RITA or nutlin treatment was examined by qPCR. Data are presented as mean±S.D., n =3. ( c and d ) MCF-7 cells were pretreated with nutlin ( c ) or RITA ( d ) followed by incubation with cycloheximide (CHX). Cells were collected at indicated time points after CHX treatment. Sp1 levels at different time points were measured by immunoblotting. ( e ) MCF-7 cells were pretreated with proteasome inhibitor MG132 followed by RITA or nutlin treatment, and Sp1 level was assessed by immunoblotting
    Figure Legend Snippet: Nutlin decreases Sp1 protein stability in p53-independent manner. ( a ) Protein levels of MDM2, Sp1 and p53 in MCF-7, HCT116 and HCT116 TP53−/− cells with RITA or nutlin treatment were assessed by immunoblotting. ( b ) mRNA level of Sp1 in MCF-7 cells with or without Sp1 depletion on RITA or nutlin treatment was examined by qPCR. Data are presented as mean±S.D., n =3. ( c and d ) MCF-7 cells were pretreated with nutlin ( c ) or RITA ( d ) followed by incubation with cycloheximide (CHX). Cells were collected at indicated time points after CHX treatment. Sp1 levels at different time points were measured by immunoblotting. ( e ) MCF-7 cells were pretreated with proteasome inhibitor MG132 followed by RITA or nutlin treatment, and Sp1 level was assessed by immunoblotting

    Techniques Used: Real-time Polymerase Chain Reaction, Incubation

    Sp1 is a key determinant of p53-mediated apoptosis, but not cell cycle arrest. ( a ) Left panel, the depletion of Sp1 in MCF-7 and HCT116 cells with two shRNAs targeting different sequences of Sp1 was assessed by immunoblotting. Actin was used as loading control. The depletion of Sp1 reduces apoptosis induced by p53-activating compounds after 48 h in MCF-7 (middle panel) and HCT116 (right panel) cells as assessed by FACS performed as in Figure 1a . ( b ) Right panel, ectopic expression of Sp1 promotes apoptosis induced by nutlin in MCF-7 and HCT116 cells but not in HCT116 TP53−/− cells as assessed by FACS performed as in Figure 1a . Left panel, the efficiency of ectopic expression of Sp1 was assessed by immunoblotting. ( c and d ) Cell cycle distribution was not affected by Sp1 depletion in MCF-7 and HCT116 cells exposed to RITA ( c ) or nutlin ( d ) as assessed by FACS performed as in Figure 1c . FACS data are presented as mean±S.D., n =3
    Figure Legend Snippet: Sp1 is a key determinant of p53-mediated apoptosis, but not cell cycle arrest. ( a ) Left panel, the depletion of Sp1 in MCF-7 and HCT116 cells with two shRNAs targeting different sequences of Sp1 was assessed by immunoblotting. Actin was used as loading control. The depletion of Sp1 reduces apoptosis induced by p53-activating compounds after 48 h in MCF-7 (middle panel) and HCT116 (right panel) cells as assessed by FACS performed as in Figure 1a . ( b ) Right panel, ectopic expression of Sp1 promotes apoptosis induced by nutlin in MCF-7 and HCT116 cells but not in HCT116 TP53−/− cells as assessed by FACS performed as in Figure 1a . Left panel, the efficiency of ectopic expression of Sp1 was assessed by immunoblotting. ( c and d ) Cell cycle distribution was not affected by Sp1 depletion in MCF-7 and HCT116 cells exposed to RITA ( c ) or nutlin ( d ) as assessed by FACS performed as in Figure 1c . FACS data are presented as mean±S.D., n =3

    Techniques Used: FACS, Expressing

    Model depicting the contribution of Sp1 to p53-mediated pro-apoptotic transcriptional repression and apoptosis. See Discussion for details
    Figure Legend Snippet: Model depicting the contribution of Sp1 to p53-mediated pro-apoptotic transcriptional repression and apoptosis. See Discussion for details

    Techniques Used:

    Sp1 is indispensable for the pro-apoptotic transcriptional repression by p53. ( a ) Heatmap representation of the genome-wide gene expression profiles (left panel) and the expression profiles of pro-apoptotic genes (right panel) in MCF-7 cells with or without Sp1 depletion on RITA or nutlin treatment. Columns indicate arrays and rows indicate genes. ( b ) Venn diagram shows the intersection of genes significantly changed by RITA treatment in MCF-7 cells with or without Sp1 depletion. P
    Figure Legend Snippet: Sp1 is indispensable for the pro-apoptotic transcriptional repression by p53. ( a ) Heatmap representation of the genome-wide gene expression profiles (left panel) and the expression profiles of pro-apoptotic genes (right panel) in MCF-7 cells with or without Sp1 depletion on RITA or nutlin treatment. Columns indicate arrays and rows indicate genes. ( b ) Venn diagram shows the intersection of genes significantly changed by RITA treatment in MCF-7 cells with or without Sp1 depletion. P

    Techniques Used: Genome Wide, Expressing

    Genome-wide shRNA screen for identifying modulators of p53-mediated apoptosis. ( a and b ) Induction of apoptosis by RITA or nutlin in MCF-7, HCT116 and HCT116 TP53−/− cells, as assessed by FACS of Rho123-PI-stained cells. Apoptosis was assayed also by FACS of Annexin V-PI-stained cells or sub-G1 population detection using PI staining. In each experiment, at least two methods were used, and conclusions were only made when similar results had been obtained with both methods. Detailed results are shown in Supplementary Figure S1B . ( b ) Induction of apoptosis by RITA was halted by blocking the p53 transcriptional activity with PFT- α , 18 but not by blocking the cytoplasmic function of p53 with PFT- μ . 19 ( c ) Effect of RITA and nutlin on cell cycle in MCF-7, HCT116 and HCT116 TP53−/− cells was assessed by FACS using PI staining. ( d ) Schematic representation showing the design of the genome-wide shRNA screen in MCF-7 cells. ( e ) Hierarchical clustering analysis of the shRNA screen data identifies two groups of hits: SLNs and RNs. Light blue indicates low abundance of shRNA (SLNs) and dark blue reflects high abundance (RNs). Rows indicate shRNAs. Raw data were normalized within each shRNA. P
    Figure Legend Snippet: Genome-wide shRNA screen for identifying modulators of p53-mediated apoptosis. ( a and b ) Induction of apoptosis by RITA or nutlin in MCF-7, HCT116 and HCT116 TP53−/− cells, as assessed by FACS of Rho123-PI-stained cells. Apoptosis was assayed also by FACS of Annexin V-PI-stained cells or sub-G1 population detection using PI staining. In each experiment, at least two methods were used, and conclusions were only made when similar results had been obtained with both methods. Detailed results are shown in Supplementary Figure S1B . ( b ) Induction of apoptosis by RITA was halted by blocking the p53 transcriptional activity with PFT- α , 18 but not by blocking the cytoplasmic function of p53 with PFT- μ . 19 ( c ) Effect of RITA and nutlin on cell cycle in MCF-7, HCT116 and HCT116 TP53−/− cells was assessed by FACS using PI staining. ( d ) Schematic representation showing the design of the genome-wide shRNA screen in MCF-7 cells. ( e ) Hierarchical clustering analysis of the shRNA screen data identifies two groups of hits: SLNs and RNs. Light blue indicates low abundance of shRNA (SLNs) and dark blue reflects high abundance (RNs). Rows indicate shRNAs. Raw data were normalized within each shRNA. P

    Techniques Used: Genome Wide, shRNA, FACS, Staining, Blocking Assay, Activity Assay

    MDM2 is required for the repression of Sp1 by nutlin. ( a ) MEFs were treated with pooled siRNA targeting p53. Protein levels of MDM2, Sp1 and p53 in wild-type MEFs and MDM2−/−TP53−/− MEFs treated with nutlin were detected by immunoblotting. GAPDH was used as loading control. ( b ) HCT116 p53-null cells were treated with pooled siRNA targeting MDM2. Protein levels of MDM2 and Sp1 in cells treated with nutlin were detected by immunoblotting. ( c ) Levels of MDM2 and Sp1 in MCF-7 cells with overexpression of Sp1 or MDM2 were assessed by immunoblotting. ( d ) The amount of MDM2 bound to Sp1 was detected by co-immunoprecipitation followed by immunoblotting. Right panel: equal amount of immunoprecipitated Sp1 was loaded to assess bound MDM2. ( e ) MCF-7, HCT116 and HCT116 TP53−/− cells were pretreated with pooled siRNA targeting MDM2. The level of ubiquitinated Sp1, Sp1 and MDM2 proteins was assessed in cells treated with proteasome inhibitor MG132 and nutlin by immunoblotting with anti-Sp1 and anti-MDM2 antibodies. ( f ) HCT116 TP53−/− cells expressing ectopic HA-tagged ubiquitin were pretreated with pooled siRNA targeting MDM2. The amount of HA-tagged ubiquitin attached to Sp1 was detected by the immunoprecipitation of Sp1 followed by the immunoblotting with anti-HA antibody. Actin was used as loading control. ( g ) Levels of MDM2, Sp1 and p53 in MCF-7 cells and Sp1-depleted MCF-7 cells on RITA or nutlin treatment were assessed by immunoblotting
    Figure Legend Snippet: MDM2 is required for the repression of Sp1 by nutlin. ( a ) MEFs were treated with pooled siRNA targeting p53. Protein levels of MDM2, Sp1 and p53 in wild-type MEFs and MDM2−/−TP53−/− MEFs treated with nutlin were detected by immunoblotting. GAPDH was used as loading control. ( b ) HCT116 p53-null cells were treated with pooled siRNA targeting MDM2. Protein levels of MDM2 and Sp1 in cells treated with nutlin were detected by immunoblotting. ( c ) Levels of MDM2 and Sp1 in MCF-7 cells with overexpression of Sp1 or MDM2 were assessed by immunoblotting. ( d ) The amount of MDM2 bound to Sp1 was detected by co-immunoprecipitation followed by immunoblotting. Right panel: equal amount of immunoprecipitated Sp1 was loaded to assess bound MDM2. ( e ) MCF-7, HCT116 and HCT116 TP53−/− cells were pretreated with pooled siRNA targeting MDM2. The level of ubiquitinated Sp1, Sp1 and MDM2 proteins was assessed in cells treated with proteasome inhibitor MG132 and nutlin by immunoblotting with anti-Sp1 and anti-MDM2 antibodies. ( f ) HCT116 TP53−/− cells expressing ectopic HA-tagged ubiquitin were pretreated with pooled siRNA targeting MDM2. The amount of HA-tagged ubiquitin attached to Sp1 was detected by the immunoprecipitation of Sp1 followed by the immunoblotting with anti-HA antibody. Actin was used as loading control. ( g ) Levels of MDM2, Sp1 and p53 in MCF-7 cells and Sp1-depleted MCF-7 cells on RITA or nutlin treatment were assessed by immunoblotting

    Techniques Used: Over Expression, Immunoprecipitation, Expressing

    11) Product Images from "CD95 Is Part of a Let-7/p53/miR-34 Regulatory Network"

    Article Title: CD95 Is Part of a Let-7/p53/miR-34 Regulatory Network

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0049636

    miR-34a and CD95 are p53 transcriptional targets that are functionally connected. ( A ) Western blot analysis of p53 and CD95 in HCT116 parental wild type (wt) and p53 −/− cells. For quantitative real-time PCR analysis of miR-34a, the same cells were treated with either control medium (−) or 10 µM etoposide (+) for 12 hrs. ( B ) MCF7, CAKI-1, and HCT116 cells were treated with control medium (−) or 10 µM etoposide (+) for 12 hrs and subjected to Western blot analysis. Band intensities were quantified relative to actin for each lane. ( C ) Apoptosis of HCT116 wt and p53 −/− cells upon LzCD95L treatment (1 µg/ml) for 18 hrs. ( D ) Key p53 targets positively correlating with the sensitivity of NCI60 cells to CD95-mediated apoptosis. ( E ) Genes whose expression positively correlates with p53 responsiveness in NCI60 cells.
    Figure Legend Snippet: miR-34a and CD95 are p53 transcriptional targets that are functionally connected. ( A ) Western blot analysis of p53 and CD95 in HCT116 parental wild type (wt) and p53 −/− cells. For quantitative real-time PCR analysis of miR-34a, the same cells were treated with either control medium (−) or 10 µM etoposide (+) for 12 hrs. ( B ) MCF7, CAKI-1, and HCT116 cells were treated with control medium (−) or 10 µM etoposide (+) for 12 hrs and subjected to Western blot analysis. Band intensities were quantified relative to actin for each lane. ( C ) Apoptosis of HCT116 wt and p53 −/− cells upon LzCD95L treatment (1 µg/ml) for 18 hrs. ( D ) Key p53 targets positively correlating with the sensitivity of NCI60 cells to CD95-mediated apoptosis. ( E ) Genes whose expression positively correlates with p53 responsiveness in NCI60 cells.

    Techniques Used: Western Blot, Real-time Polymerase Chain Reaction, Expressing

    miR-34a and let-7 are functional opposites of p53-responsiveness. ( A ) Identification of the top 10 miRNAs differentially expressed between p53 responsive and unresponsive cells. All let-7 family members are highlighted in yellow and miR-34a in light blue. ( B ) Quantitative real-time PCR analysis of miR-34a, miR-34b, and miR-34c in MCF7 cells expressing either empty vector (vec) or CD95 (CD95) after 12 hr treatment with 10 µM etoposide.
    Figure Legend Snippet: miR-34a and let-7 are functional opposites of p53-responsiveness. ( A ) Identification of the top 10 miRNAs differentially expressed between p53 responsive and unresponsive cells. All let-7 family members are highlighted in yellow and miR-34a in light blue. ( B ) Quantitative real-time PCR analysis of miR-34a, miR-34b, and miR-34c in MCF7 cells expressing either empty vector (vec) or CD95 (CD95) after 12 hr treatment with 10 µM etoposide.

    Techniques Used: Functional Assay, Real-time Polymerase Chain Reaction, Expressing, Plasmid Preparation

    CD95 positively affects p53 expression. ( A ) Left panel: MCF7 parental, vector or CD95 expressing cells treated with 10 µM etoposide for 12 hrs analyzed by western blotting for CD95, p53, and p21 expression. Right panel: p21 mRNA upregulation after 12 hrs of 20 µM etoposide treatment in vector or CD95 transfected cells. ( B ) Left panel: Effect of CD95 knockdown with a lentiviral shRNA (shR#6) on etoposide induction in CAKI-1 cells. Right panel: Effect of CD95 knockdown on etoposide induction in HCT116 cells. In both panels cells treated with 10 µM etoposide for 12 hrs. Band intensities were quantified relative to actin for each lane. ( C ) miR-34a (upper panel) and p21 (lower panel) expression analysis by quantitative real-time PCR in CAKI-1 vector or CD95 knockdown cells using shR#6 after a 12 hrs treatment with of etoposide (10 µM).
    Figure Legend Snippet: CD95 positively affects p53 expression. ( A ) Left panel: MCF7 parental, vector or CD95 expressing cells treated with 10 µM etoposide for 12 hrs analyzed by western blotting for CD95, p53, and p21 expression. Right panel: p21 mRNA upregulation after 12 hrs of 20 µM etoposide treatment in vector or CD95 transfected cells. ( B ) Left panel: Effect of CD95 knockdown with a lentiviral shRNA (shR#6) on etoposide induction in CAKI-1 cells. Right panel: Effect of CD95 knockdown on etoposide induction in HCT116 cells. In both panels cells treated with 10 µM etoposide for 12 hrs. Band intensities were quantified relative to actin for each lane. ( C ) miR-34a (upper panel) and p21 (lower panel) expression analysis by quantitative real-time PCR in CAKI-1 vector or CD95 knockdown cells using shR#6 after a 12 hrs treatment with of etoposide (10 µM).

    Techniques Used: Expressing, Plasmid Preparation, Western Blot, Transfection, shRNA, Real-time Polymerase Chain Reaction

    Model of proposed regulatory network. The three signaling branches of p53 leading to opposing outcomes of p53 activation. Experiments in this paper suggest different activation thresholds for p21 and miR-34a upon altering CD95 expression as p21 was equally efficiently induced despite modulation in CD95, whereas miR-34a was responsive to CD95 changes. Nonapoptotic activities of CD95 mentioned in this figure were discussed in recent reviews [77] , [78] .
    Figure Legend Snippet: Model of proposed regulatory network. The three signaling branches of p53 leading to opposing outcomes of p53 activation. Experiments in this paper suggest different activation thresholds for p21 and miR-34a upon altering CD95 expression as p21 was equally efficiently induced despite modulation in CD95, whereas miR-34a was responsive to CD95 changes. Nonapoptotic activities of CD95 mentioned in this figure were discussed in recent reviews [77] , [78] .

    Techniques Used: Activation Assay, Expressing

    CD95 is a negative regulator of let-7. ( A ) Let-7d expression in apoptosis resistant Type I and Type II cells among the NCI60 cell lines. ( B ) Let-7c expression determined by quantitative real-time PCR in cells with modulated CD95 expression; left panel: MCF7 cells overexpressing CD95; center panel: CAKI-1 cells with CD95 knockdown with shR#6; right panel (left): HCT116 wt cells expressing either vector or shR#6, and (right) wt and p53 −/− cells.
    Figure Legend Snippet: CD95 is a negative regulator of let-7. ( A ) Let-7d expression in apoptosis resistant Type I and Type II cells among the NCI60 cell lines. ( B ) Let-7c expression determined by quantitative real-time PCR in cells with modulated CD95 expression; left panel: MCF7 cells overexpressing CD95; center panel: CAKI-1 cells with CD95 knockdown with shR#6; right panel (left): HCT116 wt cells expressing either vector or shR#6, and (right) wt and p53 −/− cells.

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Plasmid Preparation

    12) Product Images from "Prepubertal exposure to elevated manganese results in estradiol regulated mammary gland ductal differentiation and hyperplasia in female rats"

    Article Title: Prepubertal exposure to elevated manganese results in estradiol regulated mammary gland ductal differentiation and hyperplasia in female rats

    Journal: Experimental biology and medicine (Maywood, N.J.)

    doi: 10.1177/1535370214531865

    Effects of Mn on MG protein expression of ERa, AP2a, and p53 at PND30 and 120. (a) Representative immunoblots showing expression of ERα, AP2α, and p53 proteins in MGs from saline (lanes 1–3) and Mn-treated (lanes 4–6) rats
    Figure Legend Snippet: Effects of Mn on MG protein expression of ERa, AP2a, and p53 at PND30 and 120. (a) Representative immunoblots showing expression of ERα, AP2α, and p53 proteins in MGs from saline (lanes 1–3) and Mn-treated (lanes 4–6) rats

    Techniques Used: Expressing, Western Blot

    13) Product Images from "MicroRNA-216a suppresses the proliferation and migration of human breast cancer cells via the Wnt/β-catenin signaling pathway"

    Article Title: MicroRNA-216a suppresses the proliferation and migration of human breast cancer cells via the Wnt/β-catenin signaling pathway

    Journal: Oncology Reports

    doi: 10.3892/or.2019.7050

    Anti-microRNA-216a suppresses Bax and p53 protein expression of MCF-7 cells. (A and B) Bax and p53 protein expression assessed by statistical analysis and (C) western blot analysis for Bax and p53 protein expression of MCF-7 cells. Negative, negative control group; anti-miR-216a, microRNA-216a-downregulated group. ## P
    Figure Legend Snippet: Anti-microRNA-216a suppresses Bax and p53 protein expression of MCF-7 cells. (A and B) Bax and p53 protein expression assessed by statistical analysis and (C) western blot analysis for Bax and p53 protein expression of MCF-7 cells. Negative, negative control group; anti-miR-216a, microRNA-216a-downregulated group. ## P

    Techniques Used: Expressing, Western Blot, Negative Control

    Inactivation of the Wnt pathway promotes Bax and p53 protein expression in MCF-7 cells following microRNA-216a treatment. (A and B) Bax and p53 protein expression by statistical analysis and (C) western blot analysis for Bax and p53 protein expression of MCF-7 cells. Negative, negative control group; miR-216a, microRNA-216a overexpression group; ## P
    Figure Legend Snippet: Inactivation of the Wnt pathway promotes Bax and p53 protein expression in MCF-7 cells following microRNA-216a treatment. (A and B) Bax and p53 protein expression by statistical analysis and (C) western blot analysis for Bax and p53 protein expression of MCF-7 cells. Negative, negative control group; miR-216a, microRNA-216a overexpression group; ## P

    Techniques Used: Expressing, Western Blot, Negative Control, Over Expression

    Overexpression of microRNA-216a promotes Bax and p53 protein expression of MCF-7 cells. (A and B) Bax and p53 protein expression assessed by statistical analysis and (C) western blot analysis for Bax and p53 protein expression of MCF-7 cells. Negative, negative control group; miR-216a, microRNA-216a overexpression group. ## P
    Figure Legend Snippet: Overexpression of microRNA-216a promotes Bax and p53 protein expression of MCF-7 cells. (A and B) Bax and p53 protein expression assessed by statistical analysis and (C) western blot analysis for Bax and p53 protein expression of MCF-7 cells. Negative, negative control group; miR-216a, microRNA-216a overexpression group. ## P

    Techniques Used: Over Expression, Expressing, Western Blot, Negative Control

    14) Product Images from "Actin Polymerization Negatively Regulates p53 Function by Impairing Its Nuclear Import in Response to DNA Damage"

    Article Title: Actin Polymerization Negatively Regulates p53 Function by Impairing Its Nuclear Import in Response to DNA Damage

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0060179

    Actin polymerization modulates p53 cellular accumulation. A . U2OS cells were treated with ETO (10 µM) or untreated as control for 24 h, cells were harvested and the whole cell proteins were extracted for western blotting to measure p53 protein levels. B. U2OS cells were treated with ETO (10 µM) or untreated as control for 24 h, the intensity of FITC staining p53 was measured with Image Pro Plus software. Scale bar, 10 µm. C . U2OS cells were treated with ETO (10 µM) or untreated as control for 24 h, and fluorescence assays were performed with a fluorescence microplate reader to measure cellular p53 levels (FITC intensity/DAPI intensity). D . U2OS cells were transfected with HA-actin or pcDNA, then treated with ETO (10 µM) or untreated as control at indicated time points. The cells were harvested, RNA extraction and Real Time-PCR were carried out. The mRNA content of p53 was normalized to that of GAPDH and the normal cells’ mRNA level was valued as 1. Data (mean±SD) were from three independent experiments. E . U2OS cells transfected with HA-actin were treated with ETO (10 µM) or untreated as control for 12 h. Whole cell proteins were extracted and western blotting was performed. F . U2OS cells were treated with Jas (50 nM) or CD (0.01 µg/ml) for 2 h to change actin dynamics, then treated with ETO or not. Cells were harvested, and whole cell proteins were extracted for western blotting to detect p53 protein levels in different conditions (a). Results were analyzed with Image J software (b). All Statistical differences were determined by One-way ANOVA. Jas, Jasplakinolide; CD, Cytochalasin D.
    Figure Legend Snippet: Actin polymerization modulates p53 cellular accumulation. A . U2OS cells were treated with ETO (10 µM) or untreated as control for 24 h, cells were harvested and the whole cell proteins were extracted for western blotting to measure p53 protein levels. B. U2OS cells were treated with ETO (10 µM) or untreated as control for 24 h, the intensity of FITC staining p53 was measured with Image Pro Plus software. Scale bar, 10 µm. C . U2OS cells were treated with ETO (10 µM) or untreated as control for 24 h, and fluorescence assays were performed with a fluorescence microplate reader to measure cellular p53 levels (FITC intensity/DAPI intensity). D . U2OS cells were transfected with HA-actin or pcDNA, then treated with ETO (10 µM) or untreated as control at indicated time points. The cells were harvested, RNA extraction and Real Time-PCR were carried out. The mRNA content of p53 was normalized to that of GAPDH and the normal cells’ mRNA level was valued as 1. Data (mean±SD) were from three independent experiments. E . U2OS cells transfected with HA-actin were treated with ETO (10 µM) or untreated as control for 12 h. Whole cell proteins were extracted and western blotting was performed. F . U2OS cells were treated with Jas (50 nM) or CD (0.01 µg/ml) for 2 h to change actin dynamics, then treated with ETO or not. Cells were harvested, and whole cell proteins were extracted for western blotting to detect p53 protein levels in different conditions (a). Results were analyzed with Image J software (b). All Statistical differences were determined by One-way ANOVA. Jas, Jasplakinolide; CD, Cytochalasin D.

    Techniques Used: Western Blot, Staining, Software, Fluorescence, Transfection, RNA Extraction, Real-time Polymerase Chain Reaction

    The impact of actin polymerization on p53 leads to the alteration of p21 expression. A . Cells were transfected with HA-actin or treated with Jas (50 nM) or CD (0.01 µg/ml) for 2 h. Then, cells were treated with ETO (10 µM) or untreated as control for 12 h. Whole cell proteins were extracted and western blotting was performed to measure p21 protein levels. B. Western blotting were analyzed with Image J software, and the results are presented as mean ± SD of values from three independent experiments. C. Cells were transfected with HA-actin or treated with Jas (50 nM) or CD (0.01 µg/ml) for 2 h. Then, cells were treated with ETO (10 µM) or untreated as control for 12 h. Real-Time PCR was performed, mRNA content of p21 was normalized to that of GAPDH and the normal cells’ mRNA level was valued as 1. Data (mean±SD) were from three independent experiments. All Statistical differences were determined by One-way ANOVA. **, P
    Figure Legend Snippet: The impact of actin polymerization on p53 leads to the alteration of p21 expression. A . Cells were transfected with HA-actin or treated with Jas (50 nM) or CD (0.01 µg/ml) for 2 h. Then, cells were treated with ETO (10 µM) or untreated as control for 12 h. Whole cell proteins were extracted and western blotting was performed to measure p21 protein levels. B. Western blotting were analyzed with Image J software, and the results are presented as mean ± SD of values from three independent experiments. C. Cells were transfected with HA-actin or treated with Jas (50 nM) or CD (0.01 µg/ml) for 2 h. Then, cells were treated with ETO (10 µM) or untreated as control for 12 h. Real-Time PCR was performed, mRNA content of p21 was normalized to that of GAPDH and the normal cells’ mRNA level was valued as 1. Data (mean±SD) were from three independent experiments. All Statistical differences were determined by One-way ANOVA. **, P

    Techniques Used: Expressing, Transfection, Western Blot, Software, Real-time Polymerase Chain Reaction

    Actin polymerization impairs p53 nuclear import. A. Cells were transfected with siRNA-cofilin or treated with Jas (50 nM) or CD (0.01 µg/ml) for 2 h before YFP-p53 transfection. 24 h after YFP-p53 transfection, cells were treated with ETO (10 µM) or untreated for another 12 h. Cells were then analyzed by confocalmicroscopy. Scale bar, 10 µm. B. Fluorescence values of more than 100 cells were calculated using Image J software. Results are presented as means ± SD of values from three independent experiments. C . Cells were transfected with control siRNA or cofilin siRNA, whole cell protein extraction and western blotting were then carried out to detect cofilin suppression efficiency. Panels of western blotting were analyzed with Image J software. Results are presented as means ± SD of values from three independent experiments. D. Cells were transfected with siRNA-cofilin or treated with Jas (50 nM) or CD (0.01µg/ml) for 2 h before cells were treated with ETO (10 µM) or untreated as control for another 12 h. Cytoplasmic protein and nuclear proteins were then extracted, and western blotting was performed. Oct-1 (octamer transcription factor 1) was used as nuclear protein marker and GAPDH was used as cytoplasmic protein marker (a and c). Panels of western blotting were analyzed with Image J software. Results are presented as means ± SD of values from three independent experiments (b and d). All Statistical differences were determined by One-way ANOVA. *, P
    Figure Legend Snippet: Actin polymerization impairs p53 nuclear import. A. Cells were transfected with siRNA-cofilin or treated with Jas (50 nM) or CD (0.01 µg/ml) for 2 h before YFP-p53 transfection. 24 h after YFP-p53 transfection, cells were treated with ETO (10 µM) or untreated for another 12 h. Cells were then analyzed by confocalmicroscopy. Scale bar, 10 µm. B. Fluorescence values of more than 100 cells were calculated using Image J software. Results are presented as means ± SD of values from three independent experiments. C . Cells were transfected with control siRNA or cofilin siRNA, whole cell protein extraction and western blotting were then carried out to detect cofilin suppression efficiency. Panels of western blotting were analyzed with Image J software. Results are presented as means ± SD of values from three independent experiments. D. Cells were transfected with siRNA-cofilin or treated with Jas (50 nM) or CD (0.01µg/ml) for 2 h before cells were treated with ETO (10 µM) or untreated as control for another 12 h. Cytoplasmic protein and nuclear proteins were then extracted, and western blotting was performed. Oct-1 (octamer transcription factor 1) was used as nuclear protein marker and GAPDH was used as cytoplasmic protein marker (a and c). Panels of western blotting were analyzed with Image J software. Results are presented as means ± SD of values from three independent experiments (b and d). All Statistical differences were determined by One-way ANOVA. *, P

    Techniques Used: Transfection, Fluorescence, Software, Protein Extraction, Western Blot, Marker

    Actin polymerization affects the Ser315 phosphorylation and ubiquitination of p53. A. U2OS cells transfected with HA-actin were treated with ETO (10 µM) or untreated as control for 12 h. The cells were then harvested and whole cell proteins were extracted for western blottings with indicated antibodies. B. Western blottings were analyzed using Image J software. Results are presented as mean ± SD of values from three independent experiments. C. U2OS cells transfected with HA-actin. At 24 h after the transfection, cells were treated with or without ETO for 12 h, followed by the treatment of 20 µM MG132 for 6 h. Whole cell extracts were immunoprecipitated with anti-p53 (DO-1) antibody and analyzed by western blotting with anti-p53 (FL-393) antibody. D . Cells were transfected with Aurora kinase A siRNA, and 24 h after transfection, cells were treated with ETO (10 µM) or untreated as control for 12 h. Then, cells were harvested and whole cell proteins were extracted for western blotting (a). Western blottings were analyzed using Image J software. Results are presented as mean ± SD of values from three independent experiments (b). E . Cells were harvested and subjected to immunoprecipitations using anti-Aurora A antibody and analyzed by western blotting. F . Cells were transfected with actin or actin siRNA, or treated with ETO (10 µM, 12 hours) or untreated as control, or CD (0.01µg/ml) and Jas (50 nM) for 2 h. Cells were then harvested and subjected to immunoprecipitations using anti-p53 antibody and analyzed by western blotting (a). Western blotting results were analyzed using Image J software. Results are presented as mean ± SD of values from three independent experiments (b). All Statistical differences were determined by One-way ANOVA. **, P
    Figure Legend Snippet: Actin polymerization affects the Ser315 phosphorylation and ubiquitination of p53. A. U2OS cells transfected with HA-actin were treated with ETO (10 µM) or untreated as control for 12 h. The cells were then harvested and whole cell proteins were extracted for western blottings with indicated antibodies. B. Western blottings were analyzed using Image J software. Results are presented as mean ± SD of values from three independent experiments. C. U2OS cells transfected with HA-actin. At 24 h after the transfection, cells were treated with or without ETO for 12 h, followed by the treatment of 20 µM MG132 for 6 h. Whole cell extracts were immunoprecipitated with anti-p53 (DO-1) antibody and analyzed by western blotting with anti-p53 (FL-393) antibody. D . Cells were transfected with Aurora kinase A siRNA, and 24 h after transfection, cells were treated with ETO (10 µM) or untreated as control for 12 h. Then, cells were harvested and whole cell proteins were extracted for western blotting (a). Western blottings were analyzed using Image J software. Results are presented as mean ± SD of values from three independent experiments (b). E . Cells were harvested and subjected to immunoprecipitations using anti-Aurora A antibody and analyzed by western blotting. F . Cells were transfected with actin or actin siRNA, or treated with ETO (10 µM, 12 hours) or untreated as control, or CD (0.01µg/ml) and Jas (50 nM) for 2 h. Cells were then harvested and subjected to immunoprecipitations using anti-p53 antibody and analyzed by western blotting (a). Western blotting results were analyzed using Image J software. Results are presented as mean ± SD of values from three independent experiments (b). All Statistical differences were determined by One-way ANOVA. **, P

    Techniques Used: Transfection, Western Blot, Software, Immunoprecipitation

    p53 binds to polymeric actin in the cytoplasm. A. U2OS cells were treated with Jas (50 nM) or CD (0.01 µg/ml) for 2 h and followed with ETO (10 µM) treatment for another 12 h. The cells were then harvested and subjected to immunoprecipitations using anti-p53 antibody and analyzed by western blotting (a). Western blottings of immunoprecipitated actin were analyzed using Image J software. Results are presented as means ± SD of values from three independent experiments (b). B. U2OS cells were treated with Jas (50 nM) or CD (0.01 µg/ml) for 2 h and treated with or without ETO (10 µM) for another 12 h. The cells were then harvested and the whole cell extracts were incubated with GST or recombinant GST-p53. The bound proteins were analyzed by western blotting with anti-actin antibody (top panel). GST and GST–p53 were stained with Coomassie Blue (middle panel). Arrows show the position of GST and GST–p53. Whole cell extracts were immunoblotted with antibody against GAPDH to confirm equal loading (bottom panel) (a). Western blottings of actin were analyzed using Image J software. Results are presented as means ± SD of values from three independent experiments (b). C .U2OS cells were treated with Jas (50 nM) or CD (0.01 µg/ml) for 2 h before YFP-p53 transfection. Twenty-four hours after YFP-p53 transfection, cells were treated with ETO (10 µM) for another 12 h or untreated as control and analyzed by confocalmicroscopy (a). Scale bar, 10 µm. The colocalization ratio of F-actin and YFP-p53 of more than 100 cells were calculated using Image J software. Results are presented as means ± SD of values from three independent experiments (b). All Statistical differences were determined by One-way ANOVA. *, P
    Figure Legend Snippet: p53 binds to polymeric actin in the cytoplasm. A. U2OS cells were treated with Jas (50 nM) or CD (0.01 µg/ml) for 2 h and followed with ETO (10 µM) treatment for another 12 h. The cells were then harvested and subjected to immunoprecipitations using anti-p53 antibody and analyzed by western blotting (a). Western blottings of immunoprecipitated actin were analyzed using Image J software. Results are presented as means ± SD of values from three independent experiments (b). B. U2OS cells were treated with Jas (50 nM) or CD (0.01 µg/ml) for 2 h and treated with or without ETO (10 µM) for another 12 h. The cells were then harvested and the whole cell extracts were incubated with GST or recombinant GST-p53. The bound proteins were analyzed by western blotting with anti-actin antibody (top panel). GST and GST–p53 were stained with Coomassie Blue (middle panel). Arrows show the position of GST and GST–p53. Whole cell extracts were immunoblotted with antibody against GAPDH to confirm equal loading (bottom panel) (a). Western blottings of actin were analyzed using Image J software. Results are presented as means ± SD of values from three independent experiments (b). C .U2OS cells were treated with Jas (50 nM) or CD (0.01 µg/ml) for 2 h before YFP-p53 transfection. Twenty-four hours after YFP-p53 transfection, cells were treated with ETO (10 µM) for another 12 h or untreated as control and analyzed by confocalmicroscopy (a). Scale bar, 10 µm. The colocalization ratio of F-actin and YFP-p53 of more than 100 cells were calculated using Image J software. Results are presented as means ± SD of values from three independent experiments (b). All Statistical differences were determined by One-way ANOVA. *, P

    Techniques Used: Western Blot, Immunoprecipitation, Software, Incubation, Recombinant, Staining, Transfection

    15) Product Images from "Simultaneous Inhibition of Cell-Cycle, Proliferation, Survival, Metastatic Pathways and Induction of Apoptosis in Breast Cancer Cells by a Phytochemical Super-Cocktail: Genes That Underpin Its Mode of Action"

    Article Title: Simultaneous Inhibition of Cell-Cycle, Proliferation, Survival, Metastatic Pathways and Induction of Apoptosis in Breast Cancer Cells by a Phytochemical Super-Cocktail: Genes That Underpin Its Mode of Action

    Journal: Journal of Cancer

    doi: 10.7150/jca.7235

    Molecular mechanisms of the 6-combination inhibitory actions on cell migration, invasion and induction of cell apoptosis in MCF-7 and MDA-MB-231 cell lines Cells were treated with the 6-combination for 48 hrs, protein lysates were collected and examined by western blot analysis as described under methods. All bands were quantified and normalized against β-Actin that was used as loading Control. Top panel: Synergistic down-regulation of cell proliferation marker PCNA and cell cycle regulators Rb, CDK4. Bottom panel: Down regulation of anti-apoptotic BcL-2, SVV and the cell metastatic marker cell adhesion molecule CD44 (marker of cell metastasis and BC stem cell marker) in both cell lines after 48hr from cell treatment with the 6 phytochemicals combination. Down-regulation of the MDA-MB-231 mutant P53 and up-regulation of P53 wild type in MCF7 was interestingly analyzed.
    Figure Legend Snippet: Molecular mechanisms of the 6-combination inhibitory actions on cell migration, invasion and induction of cell apoptosis in MCF-7 and MDA-MB-231 cell lines Cells were treated with the 6-combination for 48 hrs, protein lysates were collected and examined by western blot analysis as described under methods. All bands were quantified and normalized against β-Actin that was used as loading Control. Top panel: Synergistic down-regulation of cell proliferation marker PCNA and cell cycle regulators Rb, CDK4. Bottom panel: Down regulation of anti-apoptotic BcL-2, SVV and the cell metastatic marker cell adhesion molecule CD44 (marker of cell metastasis and BC stem cell marker) in both cell lines after 48hr from cell treatment with the 6 phytochemicals combination. Down-regulation of the MDA-MB-231 mutant P53 and up-regulation of P53 wild type in MCF7 was interestingly analyzed.

    Techniques Used: Migration, Multiple Displacement Amplification, Western Blot, Marker, Mutagenesis

    16) Product Images from "Downregulation of cyclin D1 sensitizes cancer cells to MDM2 antagonist Nutlin-3"

    Article Title: Downregulation of cyclin D1 sensitizes cancer cells to MDM2 antagonist Nutlin-3

    Journal: Oncotarget

    doi: 10.18632/oncotarget.8999

    Regulation of cyclin D1 expression by MDM2 ( A ) Immunoblotting evaluation of MDM2, p53, and cyclin D1 expression at 48 hours post-transfection in a mesothelioma cell line (MESO924). ( B ) Expression of MDM2, cyclin D1, p53, p21, and p27 was evaluated by immunoblotting at 96 h post-infection with MDM2 and CCND1 shRNAs in four mesothelioma cell lines (MESO924, MESO296, MESO428, and JMN1B) and a breast cancer line (MCF-7). Actin staining is a loading control. ( C ) qRT-PCR shows upregulation of CCND1 expression in three mesothelioma cell lines (MESO924, MESO296, and JMN1B) at 96 h post-infection with MDM2 shRNA2, but not TP53 . The comparative C t (cycle threshold) method was used to determine RNA expression, which was normalized to MESO257 in triplicate assays. ( D ) MDM2 expression inhibits CCND1 promoter activity: CCND1 luciferase reporter plasmid pA3−1745CD1LUC (0.25 μg), Renilla luciferase reporter plasmid pTK-RL (0.005 μg) and MDM2 or pcDNA3 empty vector (0.25 μg) were cotransfected in 293T cells. Transfected cells were harvested at 48 h, and assessed using a Dual-Luciferase Reporter Assay System. Transfection efficiencies were normalized to the pTK-RL luciferase plasmid, and CCND1 luciferase activities were normalized to the pcDNA3 vector (100%).
    Figure Legend Snippet: Regulation of cyclin D1 expression by MDM2 ( A ) Immunoblotting evaluation of MDM2, p53, and cyclin D1 expression at 48 hours post-transfection in a mesothelioma cell line (MESO924). ( B ) Expression of MDM2, cyclin D1, p53, p21, and p27 was evaluated by immunoblotting at 96 h post-infection with MDM2 and CCND1 shRNAs in four mesothelioma cell lines (MESO924, MESO296, MESO428, and JMN1B) and a breast cancer line (MCF-7). Actin staining is a loading control. ( C ) qRT-PCR shows upregulation of CCND1 expression in three mesothelioma cell lines (MESO924, MESO296, and JMN1B) at 96 h post-infection with MDM2 shRNA2, but not TP53 . The comparative C t (cycle threshold) method was used to determine RNA expression, which was normalized to MESO257 in triplicate assays. ( D ) MDM2 expression inhibits CCND1 promoter activity: CCND1 luciferase reporter plasmid pA3−1745CD1LUC (0.25 μg), Renilla luciferase reporter plasmid pTK-RL (0.005 μg) and MDM2 or pcDNA3 empty vector (0.25 μg) were cotransfected in 293T cells. Transfected cells were harvested at 48 h, and assessed using a Dual-Luciferase Reporter Assay System. Transfection efficiencies were normalized to the pTK-RL luciferase plasmid, and CCND1 luciferase activities were normalized to the pcDNA3 vector (100%).

    Techniques Used: Expressing, Transfection, Infection, Staining, Quantitative RT-PCR, RNA Expression, Activity Assay, Luciferase, Plasmid Preparation, Reporter Assay

    Immunoblotting evaluation of the effects of MDM2 inhibitor Nutlin-3 (2.5, 5, 10 μM) on expression of MDM2, p53, and cyclin D1 in mesothelioma (MESO924, MESO257, MESO296, and MESO428) and liposarcoma (LPS141 and LPS853) total cell lysates after 48 hours of treatment in serum-containing medium β-actin stain is a loading control.
    Figure Legend Snippet: Immunoblotting evaluation of the effects of MDM2 inhibitor Nutlin-3 (2.5, 5, 10 μM) on expression of MDM2, p53, and cyclin D1 in mesothelioma (MESO924, MESO257, MESO296, and MESO428) and liposarcoma (LPS141 and LPS853) total cell lysates after 48 hours of treatment in serum-containing medium β-actin stain is a loading control.

    Techniques Used: Expressing, Staining

    Additive effects were observed through coordinated inhibition of MDM2-p53 interaction and cyclin D1 as demonstrated by immunoblotting (A) and cell viability (B), showing that combination of MDM2 inhibition and cyclin D1 knockdown had greater anti-proliferative effects, compared to either intervention alone in mesothelioma cell lines (MESO924, MESO257, MESO296, MESO428, and JMN1B), a breast cancer cell line (MCF-7), a chondrosarcoma (MCS170), a liposarcoma cell line (LPS695), and a leiomyosarcoma cell line (LMS05) (A) MDM2, cyclin D1, p53, p27, p21, phospho-RB, RB, and cyclin A were evaluated by immunoblotting after treatment with Nutlin-3 for 48 hours and infection with lentiviral CCND1 shRNA for 72 hours. Actin staining is a loading control. (B) Cell viability evaluated by a Cell-titer Glo ® ATP-based luminescence assay in these cell lines, after treatment with Nutlin-3 for 48 hours and infection with lentiviral CCND1 shRNA for 72 hours. Data were normalized to the empty vector infections, DMSO, or vector and DMSO, and represent the mean values (± s.d.) from quadruplicate cultures. Statistically significant differences between DMSO and Nutlin-3, empty vector control and target gene shRNAs are presented as * p
    Figure Legend Snippet: Additive effects were observed through coordinated inhibition of MDM2-p53 interaction and cyclin D1 as demonstrated by immunoblotting (A) and cell viability (B), showing that combination of MDM2 inhibition and cyclin D1 knockdown had greater anti-proliferative effects, compared to either intervention alone in mesothelioma cell lines (MESO924, MESO257, MESO296, MESO428, and JMN1B), a breast cancer cell line (MCF-7), a chondrosarcoma (MCS170), a liposarcoma cell line (LPS695), and a leiomyosarcoma cell line (LMS05) (A) MDM2, cyclin D1, p53, p27, p21, phospho-RB, RB, and cyclin A were evaluated by immunoblotting after treatment with Nutlin-3 for 48 hours and infection with lentiviral CCND1 shRNA for 72 hours. Actin staining is a loading control. (B) Cell viability evaluated by a Cell-titer Glo ® ATP-based luminescence assay in these cell lines, after treatment with Nutlin-3 for 48 hours and infection with lentiviral CCND1 shRNA for 72 hours. Data were normalized to the empty vector infections, DMSO, or vector and DMSO, and represent the mean values (± s.d.) from quadruplicate cultures. Statistically significant differences between DMSO and Nutlin-3, empty vector control and target gene shRNAs are presented as * p

    Techniques Used: Inhibition, Infection, shRNA, Staining, Luminescence Assay, Plasmid Preparation

    Immunoblotting evaluation of MDM2, p53, and cyclin D1 expression after treatment with Nutlin-3 (2.5, 5, 10 μM) for 48 hours in mutant p53 mesothelioma cell line (JMN1B) (A) and stable p53 knockdowns at 10 days post-infection by lentiviral TP53 shRNA constructs in liposarcoma cell lines (LPS141, LPS141/239, LPS141/266 and LPS510) (B) β-actin stain is a loading control.
    Figure Legend Snippet: Immunoblotting evaluation of MDM2, p53, and cyclin D1 expression after treatment with Nutlin-3 (2.5, 5, 10 μM) for 48 hours in mutant p53 mesothelioma cell line (JMN1B) (A) and stable p53 knockdowns at 10 days post-infection by lentiviral TP53 shRNA constructs in liposarcoma cell lines (LPS141, LPS141/239, LPS141/266 and LPS510) (B) β-actin stain is a loading control.

    Techniques Used: Expressing, Mutagenesis, Infection, shRNA, Construct, Staining

    Cyclin D1 regulation by MDM2 depends on expression and ubiquitin E3-ligase activity of MDM2 ( A ) The MDM2-cyclin D1 complex was evaluated in 293T cells at 48 hours post-cotransfection of MDM2 and CCND1 by MDM2 immunoprecipitation followed by MDM2 and cyclin D1 immunoblotting. ( B ) Nutlin-3 treatment blocked the interactions of MDM2-cyclin D1 and MDM2-p53. The MDM2-cyclin D1 complex and MDM2-p53 interaction were evaluated in 293T cells at 48 hours post-cotransfection of MDM2 and CCND1 and after treatment with Nutlin-3 (5 and 10 μM) for 24 hours by MDM2 or p53 immunoprecipitation, followed by MDM2, cyclin D1 and p53 immunoblotting. ( C ) The interaction of MDM2-cyclin D1 is correlated with ubiquitin E3-ligase activity of MDM2. The MDM2-cyclin D1 complex was evaluated in 293T cells at 48 hours post-cotransfection of MDM2 and CCND1 and after treatment with Nutlin-3 (10 μM) and bortezomib (100 nM) for 24 hours by MDM2 or cyclin D1 immunoprecipitation, followed by MDM2, cyclin D1 and p53 immunoblotting. ( D ) Immunoblotting evaluation of MDM2, cyclin D1, p53, p21, p27, and ubiquitin expression after treatment with bortezomib (50 and 100 nM) and MG132 (100, 500, and 1000 nM) for 24 hours in MESO924 and MCF-7. β-actin stain is a loading control.
    Figure Legend Snippet: Cyclin D1 regulation by MDM2 depends on expression and ubiquitin E3-ligase activity of MDM2 ( A ) The MDM2-cyclin D1 complex was evaluated in 293T cells at 48 hours post-cotransfection of MDM2 and CCND1 by MDM2 immunoprecipitation followed by MDM2 and cyclin D1 immunoblotting. ( B ) Nutlin-3 treatment blocked the interactions of MDM2-cyclin D1 and MDM2-p53. The MDM2-cyclin D1 complex and MDM2-p53 interaction were evaluated in 293T cells at 48 hours post-cotransfection of MDM2 and CCND1 and after treatment with Nutlin-3 (5 and 10 μM) for 24 hours by MDM2 or p53 immunoprecipitation, followed by MDM2, cyclin D1 and p53 immunoblotting. ( C ) The interaction of MDM2-cyclin D1 is correlated with ubiquitin E3-ligase activity of MDM2. The MDM2-cyclin D1 complex was evaluated in 293T cells at 48 hours post-cotransfection of MDM2 and CCND1 and after treatment with Nutlin-3 (10 μM) and bortezomib (100 nM) for 24 hours by MDM2 or cyclin D1 immunoprecipitation, followed by MDM2, cyclin D1 and p53 immunoblotting. ( D ) Immunoblotting evaluation of MDM2, cyclin D1, p53, p21, p27, and ubiquitin expression after treatment with bortezomib (50 and 100 nM) and MG132 (100, 500, and 1000 nM) for 24 hours in MESO924 and MCF-7. β-actin stain is a loading control.

    Techniques Used: Expressing, Activity Assay, Cotransfection, Immunoprecipitation, Staining

    17) Product Images from "Fos-dependent induction of Chk1 protects osteoblasts from replication stress"

    Article Title: Fos-dependent induction of Chk1 protects osteoblasts from replication stress

    Journal: Cell Cycle

    doi: 10.4161/cc.28923

    Figure 4. FOS ameliorates RS in human OS cells in a p53-independent manner. ( A ) U2OS cells were treated with UCN-01 for 24 h, and gene expression of selected AP-1 members was assessed by qRT-PCR. Depicted are relative differences of gene expression;
    Figure Legend Snippet: Figure 4. FOS ameliorates RS in human OS cells in a p53-independent manner. ( A ) U2OS cells were treated with UCN-01 for 24 h, and gene expression of selected AP-1 members was assessed by qRT-PCR. Depicted are relative differences of gene expression;

    Techniques Used: Expressing, Quantitative RT-PCR

    18) Product Images from "Tumor-associated mutant p53 promotes cancer cell survival upon glutamine deprivation through p21 induction"

    Article Title: Tumor-associated mutant p53 promotes cancer cell survival upon glutamine deprivation through p21 induction

    Journal: Oncogene

    doi: 10.1038/onc.2016.360

    Mutp53 directly binds to the promoter of p53 target genes upon glutamine deprivation (a) CA46 cells (R248Q) were cultured in complete or Gln free medium for 16 hrs. ChIP analysis was performed to determine p53 binding to the promoter of p53 target genes in response to Gln deprivation. (b) HCT116 p53 +/+ cells or HCT116 p53 −/− cells expressing R248Q, R273H, or vector control were cultured in complete or Gln free medium for 16 hrs. ChIP analysis was performed to determine p53 binding to the promoter of p53 target genes. DNA-protein complexes were pulled down using total p53 antibody (DO-1) or isotype matched IgG. All p53 binding sites were assessed by PCR. PCR products were separated using agarose electrophoresis.
    Figure Legend Snippet: Mutp53 directly binds to the promoter of p53 target genes upon glutamine deprivation (a) CA46 cells (R248Q) were cultured in complete or Gln free medium for 16 hrs. ChIP analysis was performed to determine p53 binding to the promoter of p53 target genes in response to Gln deprivation. (b) HCT116 p53 +/+ cells or HCT116 p53 −/− cells expressing R248Q, R273H, or vector control were cultured in complete or Gln free medium for 16 hrs. ChIP analysis was performed to determine p53 binding to the promoter of p53 target genes. DNA-protein complexes were pulled down using total p53 antibody (DO-1) or isotype matched IgG. All p53 binding sites were assessed by PCR. PCR products were separated using agarose electrophoresis.

    Techniques Used: Cell Culture, Chromatin Immunoprecipitation, Binding Assay, Expressing, Plasmid Preparation, Polymerase Chain Reaction, Electrophoresis

    Tumors expressing mutant p53 are more resistant to glutaminase inhibitor treatment in vivo (a,b) Athymic Nude mice at 7 weeks old were injected with HCT116 p53 −/− cells on the left flank. HCT116 p53 −/− cells expressing mutp53 R248Q were injected on the right flank. Once the tumor size reached an average of 60 mm 3 , the mice were treated with 15 mg/kg of L-DON every other day by i.p. injection. Tumor size was measured over time. Data represent the mean ± S.D. (n=5 or 6 tumors as indicated), *** P ≤.001, Student’s t-test. (c) Tumors with L-DON or vehicle treatment were harvested at day 11. Western blot was performed using the indicated antibodies.
    Figure Legend Snippet: Tumors expressing mutant p53 are more resistant to glutaminase inhibitor treatment in vivo (a,b) Athymic Nude mice at 7 weeks old were injected with HCT116 p53 −/− cells on the left flank. HCT116 p53 −/− cells expressing mutp53 R248Q were injected on the right flank. Once the tumor size reached an average of 60 mm 3 , the mice were treated with 15 mg/kg of L-DON every other day by i.p. injection. Tumor size was measured over time. Data represent the mean ± S.D. (n=5 or 6 tumors as indicated), *** P ≤.001, Student’s t-test. (c) Tumors with L-DON or vehicle treatment were harvested at day 11. Western blot was performed using the indicated antibodies.

    Techniques Used: Expressing, Mutagenesis, In Vivo, Mouse Assay, Injection, Western Blot

    Loss of mutp53 sensitizes cancer cells to glutamine deprivation and glutaminase inhibitor treatment (a–b) p53 mRNA levels (a) relative to actin and protein levels (b) in control vector and p53 shRNA transduced CA46 cells was assessed using qRT-PCR and Western blot. (c) The indicated control vector or shRNA p53 transduced cells were cultured in complete or Gln free medium for the indicated time points. Viability was assessed by PI exclusion and normalized to cells cultured in complete medium. Data represent mean ± S.D. of three independent experiments (*** P ≤.001, Student’s t test). (d–e) Annexin-V and PI staining of stably transfected CA46 cells cultured in Gln free medium or complete medium for 24 hrs. Representative graphs of cells in late apoptosis (annexin-V and PI positive) are shown. Data represent mean ± S.D. of three independent experiments (*** P ≤.001, Student’s t test). (f) The control vector transduced cells and p53 shRNA transduced cells were treated with 12.5µM L-DON for two days, 40µM Compound 968 and 50µM BPTES for four days. Cell viability was assessed by PI exclusion and normalized to control treated cells. Data represent mean ± S.D. of three independent experiments (* P
    Figure Legend Snippet: Loss of mutp53 sensitizes cancer cells to glutamine deprivation and glutaminase inhibitor treatment (a–b) p53 mRNA levels (a) relative to actin and protein levels (b) in control vector and p53 shRNA transduced CA46 cells was assessed using qRT-PCR and Western blot. (c) The indicated control vector or shRNA p53 transduced cells were cultured in complete or Gln free medium for the indicated time points. Viability was assessed by PI exclusion and normalized to cells cultured in complete medium. Data represent mean ± S.D. of three independent experiments (*** P ≤.001, Student’s t test). (d–e) Annexin-V and PI staining of stably transfected CA46 cells cultured in Gln free medium or complete medium for 24 hrs. Representative graphs of cells in late apoptosis (annexin-V and PI positive) are shown. Data represent mean ± S.D. of three independent experiments (*** P ≤.001, Student’s t test). (f) The control vector transduced cells and p53 shRNA transduced cells were treated with 12.5µM L-DON for two days, 40µM Compound 968 and 50µM BPTES for four days. Cell viability was assessed by PI exclusion and normalized to control treated cells. Data represent mean ± S.D. of three independent experiments (* P

    Techniques Used: Plasmid Preparation, shRNA, Quantitative RT-PCR, Western Blot, Cell Culture, Staining, Stable Transfection, Transfection

    Expression of mutp53 promotes cell survival upon glutamine deprivation and glutaminase inhibitor treatment (a) Mutp53 R248Q or R273H were stably expressed in p53 −/− HCT116 cells. Protein levels were assessed by Western blot using antibodies against p53 (DO-1) and actin. (b) HCT116 p53 −/− cells expressing an empty vector or mutp53 protein were cultured in either Gln free or complete medium for the indicated time points. Cell viability was determined by Trypan blue exclusion and normalized to cells cultured in complete medium. Data represent mean ± S.E.M. of three independent experiments. (c) HCT116 p53 −/− cells expressing an empty vector or mutp53 protein were cultured in Gln free medium or complete medium for 4 days. Lysate was extracted to perform Western blot analysis using antibodies as indicated. (d) HCT116 p53 −/− cells expressing R248Q, R273H, or empty vector and HCT116 p53 +/+ cells were cultured in Gln free medium or complete medium for three days. (e) HCT116 p53 −/− cells expressing R248Q, R273H, or empty vector were treated with 50µM L-DON for four days, or 40µM Compound 968 for five days. (f) HCT116 p53 −/− cells expressing R248Q, R273H, or empty vector were treated with 5µM camptothecin (CPT) for three days, 10µM doxorubicin (DOXO) for four days, or 10 nM docetaxel (DTX) for two days. (d–f) Cell viability was determined by Trypan blue exclusion and normalized to cells cultured in complete medium. Data represent mean ± S.E.M. of three independent experiments (** P
    Figure Legend Snippet: Expression of mutp53 promotes cell survival upon glutamine deprivation and glutaminase inhibitor treatment (a) Mutp53 R248Q or R273H were stably expressed in p53 −/− HCT116 cells. Protein levels were assessed by Western blot using antibodies against p53 (DO-1) and actin. (b) HCT116 p53 −/− cells expressing an empty vector or mutp53 protein were cultured in either Gln free or complete medium for the indicated time points. Cell viability was determined by Trypan blue exclusion and normalized to cells cultured in complete medium. Data represent mean ± S.E.M. of three independent experiments. (c) HCT116 p53 −/− cells expressing an empty vector or mutp53 protein were cultured in Gln free medium or complete medium for 4 days. Lysate was extracted to perform Western blot analysis using antibodies as indicated. (d) HCT116 p53 −/− cells expressing R248Q, R273H, or empty vector and HCT116 p53 +/+ cells were cultured in Gln free medium or complete medium for three days. (e) HCT116 p53 −/− cells expressing R248Q, R273H, or empty vector were treated with 50µM L-DON for four days, or 40µM Compound 968 for five days. (f) HCT116 p53 −/− cells expressing R248Q, R273H, or empty vector were treated with 5µM camptothecin (CPT) for three days, 10µM doxorubicin (DOXO) for four days, or 10 nM docetaxel (DTX) for two days. (d–f) Cell viability was determined by Trypan blue exclusion and normalized to cells cultured in complete medium. Data represent mean ± S.E.M. of three independent experiments (** P

    Techniques Used: Expressing, Stable Transfection, Western Blot, Plasmid Preparation, Cell Culture, Cycling Probe Technology

    Mutp53 induces expression of p53 target genes upon glutamine deprivation (a) EB3 and CA46 cells were cultured in complete or Gln free medium for 24 hrs. Cells were lysed for Western blot using antibodies as indicated. (b) EB3 and CA46 cells were cultured in complete or Gln free medium overnight. mRNA expression of p53 target genes relative to 18S was determined using qRT- PCR and normalized to the complete medium. (c) Cells were transduced with lenti-viral particles followed by puromycin selection to generate stable knockdown of wtp53 in EB3 cells and mutp53 in CA46. p53 protein levels were determined by Western blot. (d) EB3 and CA46 cells infected with virus containing control vector or shRNA against p53 were cultured in Gln free medium overnight. mRNA expression of p53 target genes relative to actin was determined using qRT- PCR and normalized to the complete control medium. (e) HCT116 p53 −/− cells expressing R248Q, R273H, or empty vector were cultured in Gln free medium for three days. p53 activation and total p53 expression was determined by Western blot analysis using anti-phospho-p53 (Ser15) and anti-p53 antibody. (f) HCT116 p53 +/+ cells and HCT116 p53 −/− cells expressing R248Q, R273H, or empty vector were cultured in complete or Gln free medium overnight. mRNA expression of p53 target genes relative to actin was determined using qRT-PCR and normalized to the complete medium. Data represent mean ± S.D. of duplicates from two independent experiments (* P
    Figure Legend Snippet: Mutp53 induces expression of p53 target genes upon glutamine deprivation (a) EB3 and CA46 cells were cultured in complete or Gln free medium for 24 hrs. Cells were lysed for Western blot using antibodies as indicated. (b) EB3 and CA46 cells were cultured in complete or Gln free medium overnight. mRNA expression of p53 target genes relative to 18S was determined using qRT- PCR and normalized to the complete medium. (c) Cells were transduced with lenti-viral particles followed by puromycin selection to generate stable knockdown of wtp53 in EB3 cells and mutp53 in CA46. p53 protein levels were determined by Western blot. (d) EB3 and CA46 cells infected with virus containing control vector or shRNA against p53 were cultured in Gln free medium overnight. mRNA expression of p53 target genes relative to actin was determined using qRT- PCR and normalized to the complete control medium. (e) HCT116 p53 −/− cells expressing R248Q, R273H, or empty vector were cultured in Gln free medium for three days. p53 activation and total p53 expression was determined by Western blot analysis using anti-phospho-p53 (Ser15) and anti-p53 antibody. (f) HCT116 p53 +/+ cells and HCT116 p53 −/− cells expressing R248Q, R273H, or empty vector were cultured in complete or Gln free medium overnight. mRNA expression of p53 target genes relative to actin was determined using qRT-PCR and normalized to the complete medium. Data represent mean ± S.D. of duplicates from two independent experiments (* P

    Techniques Used: Expressing, Cell Culture, Western Blot, Quantitative RT-PCR, Transduction, Selection, Infection, Plasmid Preparation, shRNA, Activation Assay

    Mutp53 promotes cell survival upon glutamine deprivation through p21 induction (a,b) HCT116 p53 −/− cells expressing mutp53 (R248Q) or empty vector were cultured in complete or Gln free medium for 24 hrs. PI staining followed by Flow Cytometry was performed to assess cell cycle profile. Representative graphs of three independent experiments are shown. Data represent the mean ± S.D. (c) HCT116 p53 −/− cells expressing mutp53 R248Q or empty vector were cultured in complete or Gln free medium for 24 hours. Western blots were performed using antibodies as indicated. (d) p21 was transiently knocked down in HCT116 p53 −/− cells expressing mutp53 R248Q or empty vector using siRNA (20nM). 48 hrs after the siRNA transfection, cells were cultured in complete or Gln free medium for four days. Cell viability was determined by Trypan blue exclusion. Data represent the mean ± S.D. of four independent experiments, (*** P ≤.001, Student’s t-test). (e) Western blot was performed using antibodies as indicated.
    Figure Legend Snippet: Mutp53 promotes cell survival upon glutamine deprivation through p21 induction (a,b) HCT116 p53 −/− cells expressing mutp53 (R248Q) or empty vector were cultured in complete or Gln free medium for 24 hrs. PI staining followed by Flow Cytometry was performed to assess cell cycle profile. Representative graphs of three independent experiments are shown. Data represent the mean ± S.D. (c) HCT116 p53 −/− cells expressing mutp53 R248Q or empty vector were cultured in complete or Gln free medium for 24 hours. Western blots were performed using antibodies as indicated. (d) p21 was transiently knocked down in HCT116 p53 −/− cells expressing mutp53 R248Q or empty vector using siRNA (20nM). 48 hrs after the siRNA transfection, cells were cultured in complete or Gln free medium for four days. Cell viability was determined by Trypan blue exclusion. Data represent the mean ± S.D. of four independent experiments, (*** P ≤.001, Student’s t-test). (e) Western blot was performed using antibodies as indicated.

    Techniques Used: Expressing, Plasmid Preparation, Cell Culture, Staining, Flow Cytometry, Cytometry, Western Blot, Transfection

    Cancer cells with mutp53 are more resistant to glutamine deprivation than cells with wtp53 (a) Lymphoma cell lines with known p53 status were cultured in complete or glutamine (Gln) free medium for 2 days. Viability was determined by propidium iodide (PI) exclusion and normalized to cells cultured in complete medium. Mutp53 cells: CA46, SupT1, DB. Wtp53 cells: EB3, DOHH2, LY3. (b) EB3 and CA46 cells were cultured in Gln free medium for 24 hrs. Western blot was performed using antibodies as indicated. (c,d) EB3 and CA46 cells were cultured in complete or Gln free medium for 48 hrs. Apoptosis was assessed by staining cells with annexin-V and PI. Positive staining was measured by flow cytometry and analyzed with FlowJo. Representative graphs and percentage of late apoptotic cells (PI and annexin-V positive cells) are shown. Data represent mean ± S.D. of three independent experiments (*** P ≤.001, Student’s t test). (e) EB3 and CA46 cells were treated with L-DON for 3 days, Compound 968 and BPTES for 4 days. Cell viability was assessed by PI exclusion, and normalized to control treated cells. Data represents mean ± S.D. of three independent experiments (** P
    Figure Legend Snippet: Cancer cells with mutp53 are more resistant to glutamine deprivation than cells with wtp53 (a) Lymphoma cell lines with known p53 status were cultured in complete or glutamine (Gln) free medium for 2 days. Viability was determined by propidium iodide (PI) exclusion and normalized to cells cultured in complete medium. Mutp53 cells: CA46, SupT1, DB. Wtp53 cells: EB3, DOHH2, LY3. (b) EB3 and CA46 cells were cultured in Gln free medium for 24 hrs. Western blot was performed using antibodies as indicated. (c,d) EB3 and CA46 cells were cultured in complete or Gln free medium for 48 hrs. Apoptosis was assessed by staining cells with annexin-V and PI. Positive staining was measured by flow cytometry and analyzed with FlowJo. Representative graphs and percentage of late apoptotic cells (PI and annexin-V positive cells) are shown. Data represent mean ± S.D. of three independent experiments (*** P ≤.001, Student’s t test). (e) EB3 and CA46 cells were treated with L-DON for 3 days, Compound 968 and BPTES for 4 days. Cell viability was assessed by PI exclusion, and normalized to control treated cells. Data represents mean ± S.D. of three independent experiments (** P

    Techniques Used: Cell Culture, Western Blot, Staining, Flow Cytometry, Cytometry

    19) Product Images from "Genistein inhibits proliferation of colon cancer cells by attenuating a negative effect of epidermal growth factor on tumor suppressor FOXO3 activity"

    Article Title: Genistein inhibits proliferation of colon cancer cells by attenuating a negative effect of epidermal growth factor on tumor suppressor FOXO3 activity

    Journal: BMC Cancer

    doi: 10.1186/1471-2407-11-219

    The FOXO3-p53(mut) complex promotes p27kip1 expression . (A) The protein-DNA complex was immunoprecipitated with antibody against p53 and DNA was amplified by conventional PCR with primers from the p27kip1 promoter region (ChIP assay) with input representing PCR amplification from lysate before immunoprecipitation with the same primers. The FOXO3-p53(mut) complex is bound to the p27kip1 promoter. (B) HT-29 cells transfected with p53 siRNA or scramble siRNA were treated with genistein and examined for p27kip1 by immunoblot. Silencing of p53 attenuates genistein-induced p27kip1 expression. These experiments were repeated two independent times.
    Figure Legend Snippet: The FOXO3-p53(mut) complex promotes p27kip1 expression . (A) The protein-DNA complex was immunoprecipitated with antibody against p53 and DNA was amplified by conventional PCR with primers from the p27kip1 promoter region (ChIP assay) with input representing PCR amplification from lysate before immunoprecipitation with the same primers. The FOXO3-p53(mut) complex is bound to the p27kip1 promoter. (B) HT-29 cells transfected with p53 siRNA or scramble siRNA were treated with genistein and examined for p27kip1 by immunoblot. Silencing of p53 attenuates genistein-induced p27kip1 expression. These experiments were repeated two independent times.

    Techniques Used: Expressing, Immunoprecipitation, Amplification, Polymerase Chain Reaction, Chromatin Immunoprecipitation, Transfection

    Genistein increases FOXO3-p53(mut) interaction in HT-29 cells . (A) Protein from control and genistein treated HT-29 cells was immunoblotted for p53. Genistein increased p53 expression 2.5-fold. The experiment was performed three independent times and quantified using densitometry (n = 3, *p
    Figure Legend Snippet: Genistein increases FOXO3-p53(mut) interaction in HT-29 cells . (A) Protein from control and genistein treated HT-29 cells was immunoblotted for p53. Genistein increased p53 expression 2.5-fold. The experiment was performed three independent times and quantified using densitometry (n = 3, *p

    Techniques Used: Expressing

    Genistein inhibits proliferation in colon cancer cells by promoting FOXO3 activity . Schematic representation of pathways targeted by genistein in colon cancer cells [ 7 - 9 , 13 , 21 , 50 ]. Genistein promotes FOXO3 activity by inhibiting PI3K/Akt and stimulating FOXO3 interaction with p53. Downstream, genistein-mediated FOXO3 activity increases p27kip1 expression, which promotes cell cycle arrest and leads to inhibition of proliferation.
    Figure Legend Snippet: Genistein inhibits proliferation in colon cancer cells by promoting FOXO3 activity . Schematic representation of pathways targeted by genistein in colon cancer cells [ 7 - 9 , 13 , 21 , 50 ]. Genistein promotes FOXO3 activity by inhibiting PI3K/Akt and stimulating FOXO3 interaction with p53. Downstream, genistein-mediated FOXO3 activity increases p27kip1 expression, which promotes cell cycle arrest and leads to inhibition of proliferation.

    Techniques Used: Activity Assay, Expressing, Inhibition

    20) Product Images from "REDD1 Protects Osteoblast Cells from Gamma Radiation-Induced Premature Senescence"

    Article Title: REDD1 Protects Osteoblast Cells from Gamma Radiation-Induced Premature Senescence

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0036604

    IR induced REDD1 signal transduction cascades. (A) hFOB cell lysates collected at 4 h after IR were subjected to immunoprecipitation using REDD1, NFkBp65 or p53 antibodies, respectively. After SDS-gel separation, proteins were analyzed by immunoblotting using anti-REDD1, NFkBp65, RPA2 and p53 antibodies. (B) Western blot using hFOB cell lysates shows REDD1, p53, NFkBp65 and β-actin (loading control) expression in control, NFkBp65-siRNA-transfected, and p53-siRNA-transfected samples. Knockdown of either NFkBp65 or p53 gene resulted in attenuated REDD1 protein expression. (C) Western blot shows REDD1, p53 and NFkBp65 expression and p53 (serc15) and NFkBp65 (ser 536) phosphorylation in control, vector or REDD1 plasmid DNA-transfected samples. Overexpression of REDD1 had no effects on p53 and NFkB expression and activation. Representative immunoblots from three experiments are shown.
    Figure Legend Snippet: IR induced REDD1 signal transduction cascades. (A) hFOB cell lysates collected at 4 h after IR were subjected to immunoprecipitation using REDD1, NFkBp65 or p53 antibodies, respectively. After SDS-gel separation, proteins were analyzed by immunoblotting using anti-REDD1, NFkBp65, RPA2 and p53 antibodies. (B) Western blot using hFOB cell lysates shows REDD1, p53, NFkBp65 and β-actin (loading control) expression in control, NFkBp65-siRNA-transfected, and p53-siRNA-transfected samples. Knockdown of either NFkBp65 or p53 gene resulted in attenuated REDD1 protein expression. (C) Western blot shows REDD1, p53 and NFkBp65 expression and p53 (serc15) and NFkBp65 (ser 536) phosphorylation in control, vector or REDD1 plasmid DNA-transfected samples. Overexpression of REDD1 had no effects on p53 and NFkB expression and activation. Representative immunoblots from three experiments are shown.

    Techniques Used: Transduction, Immunoprecipitation, SDS-Gel, Western Blot, Expressing, Transfection, Plasmid Preparation, Over Expression, Activation Assay

    21) Product Images from "HAUSP deubiquitinated and stabilizes N-Myc in neuroblastoma"

    Article Title: HAUSP deubiquitinated and stabilizes N-Myc in neuroblastoma

    Journal: Nature medicine

    doi: 10.1038/nm.4180

    HAUSP affects and directly interacts with N-Myc both in vitro and in vivo ( a ) Western blot of mouse brains collected at day E13.5; n = 2. ( b ) Representative immunohistochemistry of HAUSP, N-Myc and p53 of E18.5 mouse cortex sections of the marginal zone to the cortical plate separated by a dotted black line; magnification 40 ×; scale bar 25 μm; n = 2 per group. ( c ) N-Myc expression vector cotransfected with Flag-HAUSP (lane 2) or empty vector (lane 1) in HEK293T cells. Cell lysates were incubated with Flag/M2 beads then subjected to western blot; 10% input; n = 3. ( d, e ) Endogenous immunoprecipitation of N-Myc with HAUSP from native IMR-32 ( d ) and SK-N-BE(2)C cells ( e ); 2% input; n = 3. ( f ) Direct interaction between purified Flag-N-Myc and GST-HAUSP. Purified N-Myc was incubated with GST protein (lane 1) or GST-HAUSP (lane 2) and immobilized with GST beads then subjected to western blot; 10% input; n = 2. ( g ) Direct interaction between purified Flag-HAUSP and GST-N-Myc as in Fig. 1f . Proteins were immobilized with GST beads then subjected to western blot; 1% input; n = 3. ( h ) Schematic representation of N-Myc deletion mutants used for domain mapping. ( i ) Indicated N-Myc expression vectors cotransfected with HAUSP in HEK293T cells. Lysates were incubated with HA beads then subjected to western blot; 10% input; n = 3.
    Figure Legend Snippet: HAUSP affects and directly interacts with N-Myc both in vitro and in vivo ( a ) Western blot of mouse brains collected at day E13.5; n = 2. ( b ) Representative immunohistochemistry of HAUSP, N-Myc and p53 of E18.5 mouse cortex sections of the marginal zone to the cortical plate separated by a dotted black line; magnification 40 ×; scale bar 25 μm; n = 2 per group. ( c ) N-Myc expression vector cotransfected with Flag-HAUSP (lane 2) or empty vector (lane 1) in HEK293T cells. Cell lysates were incubated with Flag/M2 beads then subjected to western blot; 10% input; n = 3. ( d, e ) Endogenous immunoprecipitation of N-Myc with HAUSP from native IMR-32 ( d ) and SK-N-BE(2)C cells ( e ); 2% input; n = 3. ( f ) Direct interaction between purified Flag-N-Myc and GST-HAUSP. Purified N-Myc was incubated with GST protein (lane 1) or GST-HAUSP (lane 2) and immobilized with GST beads then subjected to western blot; 10% input; n = 2. ( g ) Direct interaction between purified Flag-HAUSP and GST-N-Myc as in Fig. 1f . Proteins were immobilized with GST beads then subjected to western blot; 1% input; n = 3. ( h ) Schematic representation of N-Myc deletion mutants used for domain mapping. ( i ) Indicated N-Myc expression vectors cotransfected with HAUSP in HEK293T cells. Lysates were incubated with HA beads then subjected to western blot; 10% input; n = 3.

    Techniques Used: In Vitro, In Vivo, Western Blot, Immunohistochemistry, Expressing, Plasmid Preparation, Incubation, Immunoprecipitation, Purification

    22) Product Images from "Reversal of human cellular senescence: roles of the p53 and p16 pathways"

    Article Title: Reversal of human cellular senescence: roles of the p53 and p16 pathways

    Journal: The EMBO Journal

    doi: 10.1093/emboj/cdg417

    Fig. 2. p53 inactivation reverses senescence of BJ, but not WI-38 fibroblasts. ( A ) S-WI cells synthesize DNA, but do not proliferate. S-WI cells were infected with lentiviruses expressing GFP, hTERT, GSE, LgT, LgTK1 and CDK4m as indicated; 72 h later, DNA synthesis was determined by % LN, and percentage growth monitored, as described in the text. ( B ) S-BJ cells synthesize DNA and proliferate. S-BJ cells were infected and monitored, as described in (A). ( C ) Morphology of control and rescued S-BJ cells. S-BJ cells infected with GFP or GSE-expressing lentivirus were photographed 6 days later. ( D ) Lifespan assays. S-BJ cells were infected with lentiviruses expressing the indicated proteins, serially passaged, and cell number determined at each passage, as described in Materials and methods.
    Figure Legend Snippet: Fig. 2. p53 inactivation reverses senescence of BJ, but not WI-38 fibroblasts. ( A ) S-WI cells synthesize DNA, but do not proliferate. S-WI cells were infected with lentiviruses expressing GFP, hTERT, GSE, LgT, LgTK1 and CDK4m as indicated; 72 h later, DNA synthesis was determined by % LN, and percentage growth monitored, as described in the text. ( B ) S-BJ cells synthesize DNA and proliferate. S-BJ cells were infected and monitored, as described in (A). ( C ) Morphology of control and rescued S-BJ cells. S-BJ cells infected with GFP or GSE-expressing lentivirus were photographed 6 days later. ( D ) Lifespan assays. S-BJ cells were infected with lentiviruses expressing the indicated proteins, serially passaged, and cell number determined at each passage, as described in Materials and methods.

    Techniques Used: Infection, Expressing, DNA Synthesis

    Fig. 7. Pathways leading to reversible and essentially irreversible senescence growth arrests in human cells. Proliferating cells (Presenescent) arrest growth with a senescent phenotype in response to telomere erosion, which is p53 dependent, or a combination of telomere erosion and an as yet unidentified stimulus that induces p16. The p53-dependent arrest increases p21 expression, and is reversed by p53 inactivation or oncogenic Ras. p53 inactivation results in extensive proliferation (growth) culminating in crisis, whereas Ras causes limited proliferation. Cells that senesce with high p16 can be stimulated to synthesize DNA (S-phase) upon inactivation of p53 and pRb, or pRb inactivation plus oncogenic Ras, but do not proliferate (no growth).
    Figure Legend Snippet: Fig. 7. Pathways leading to reversible and essentially irreversible senescence growth arrests in human cells. Proliferating cells (Presenescent) arrest growth with a senescent phenotype in response to telomere erosion, which is p53 dependent, or a combination of telomere erosion and an as yet unidentified stimulus that induces p16. The p53-dependent arrest increases p21 expression, and is reversed by p53 inactivation or oncogenic Ras. p53 inactivation results in extensive proliferation (growth) culminating in crisis, whereas Ras causes limited proliferation. Cells that senesce with high p16 can be stimulated to synthesize DNA (S-phase) upon inactivation of p53 and pRb, or pRb inactivation plus oncogenic Ras, but do not proliferate (no growth).

    Techniques Used: Expressing

    23) Product Images from "TRIM59 promotes breast cancer motility by suppressing p62-selective autophagic degradation of PDCD10"

    Article Title: TRIM59 promotes breast cancer motility by suppressing p62-selective autophagic degradation of PDCD10

    Journal: PLoS Biology

    doi: 10.1371/journal.pbio.3000051

    TRIM59 interacts with PDCD10 and blocks the autophagic degradation of PDCD10. ( A ) Yeast two-hybrid screening to confirm the interaction between TRIM59-BD and PDCD10-AD. Transformed and mated cells were grown on–Leu–Trp medium ( bottom ). Interaction of TRIM59 with PDCD10 was verified on high-stringency plates (–Leu–Trp–His–Ade) and with a β-galactosidase filter assay and Aureobasidin A (AbA) ( top ). Interaction of p53 and SV40 large T-antigen (LTA) was used as positive control. ( B ) Co-IP and IB analysis of extracts of MCF7 cells with the indicated antibodies. IP with IgG was added as a negative control. ( C ) Co-IP and IB analysis of extracts of HEK293T cells transfected with FLAG-PDCD10 along with HA-tagged TRIM59 variants (full-length WT or deletions [ΔR, ΔTM, B, and CC] as shown on the top cartoon). ( D ) IB and qPCR analysis of PDCD10 protein expression (left) and mRNA levels (right) in shControl and sh TRIM59 MCF7 cells ( D , lane 1 versus 2) or in control and TRIM59 OE MDA-MB-231 cells ( D . ( E ) Representative immunofluorescent staining of PDCD10 (green) in sections from xenograft of shControl or sh TRIM59 MCF7 cells. DAPI, DNA-intercalating dye, indicates the nucleus (blue). Scale bars: 100 μm. n = 4. ( F ) IB analysis of PDCD10 expression in MCF7 WT or TRIM59 KO cell fractions. β-actin and LaminB1 were used as markers for the cytosolic and nuclear fractions, respectively. ( G for similar experiments using TRIM59 KD cells. ΔR, TRIM59 without RING domain; ΔTM, TRIM59 without the predicted transmembrane domain; AbA, Aureobasidin A; AD, transcription activation domain;–Ade, adenine dropout; B, B-box-type zinc finger domain; BafA1, bafilomycin A1; BD, DNA-binding domain; C, cytosolic fraction; CC, coiled-coil domain; co-IP, co-immunoprecipitation; HA, hemagglutinin;–His, histidine dropout; IB, immunoblot; IgG, immunoglobulin G; IP, immunoprecipitation; KD, knockdown; KO, knockout;–Leu, Leucine dropout; LTA, large T-antigen; N, nuclear fraction; NS, not significant; OE, overexpressing; PDCD10, programmed cell death protein 10; p53, tumor protein 53; qPCR, quantitative polymerase chain reaction; SV40, polyomavirus simian virus 40; TM, transmembrane domain; TRIM59, tripartite motif 59;–Trp, tryptophan dropout; WT, wild-type.
    Figure Legend Snippet: TRIM59 interacts with PDCD10 and blocks the autophagic degradation of PDCD10. ( A ) Yeast two-hybrid screening to confirm the interaction between TRIM59-BD and PDCD10-AD. Transformed and mated cells were grown on–Leu–Trp medium ( bottom ). Interaction of TRIM59 with PDCD10 was verified on high-stringency plates (–Leu–Trp–His–Ade) and with a β-galactosidase filter assay and Aureobasidin A (AbA) ( top ). Interaction of p53 and SV40 large T-antigen (LTA) was used as positive control. ( B ) Co-IP and IB analysis of extracts of MCF7 cells with the indicated antibodies. IP with IgG was added as a negative control. ( C ) Co-IP and IB analysis of extracts of HEK293T cells transfected with FLAG-PDCD10 along with HA-tagged TRIM59 variants (full-length WT or deletions [ΔR, ΔTM, B, and CC] as shown on the top cartoon). ( D ) IB and qPCR analysis of PDCD10 protein expression (left) and mRNA levels (right) in shControl and sh TRIM59 MCF7 cells ( D , lane 1 versus 2) or in control and TRIM59 OE MDA-MB-231 cells ( D . ( E ) Representative immunofluorescent staining of PDCD10 (green) in sections from xenograft of shControl or sh TRIM59 MCF7 cells. DAPI, DNA-intercalating dye, indicates the nucleus (blue). Scale bars: 100 μm. n = 4. ( F ) IB analysis of PDCD10 expression in MCF7 WT or TRIM59 KO cell fractions. β-actin and LaminB1 were used as markers for the cytosolic and nuclear fractions, respectively. ( G for similar experiments using TRIM59 KD cells. ΔR, TRIM59 without RING domain; ΔTM, TRIM59 without the predicted transmembrane domain; AbA, Aureobasidin A; AD, transcription activation domain;–Ade, adenine dropout; B, B-box-type zinc finger domain; BafA1, bafilomycin A1; BD, DNA-binding domain; C, cytosolic fraction; CC, coiled-coil domain; co-IP, co-immunoprecipitation; HA, hemagglutinin;–His, histidine dropout; IB, immunoblot; IgG, immunoglobulin G; IP, immunoprecipitation; KD, knockdown; KO, knockout;–Leu, Leucine dropout; LTA, large T-antigen; N, nuclear fraction; NS, not significant; OE, overexpressing; PDCD10, programmed cell death protein 10; p53, tumor protein 53; qPCR, quantitative polymerase chain reaction; SV40, polyomavirus simian virus 40; TM, transmembrane domain; TRIM59, tripartite motif 59;–Trp, tryptophan dropout; WT, wild-type.

    Techniques Used: Two Hybrid Screening, Transformation Assay, Positive Control, Co-Immunoprecipitation Assay, Negative Control, Transfection, Real-time Polymerase Chain Reaction, Expressing, Multiple Displacement Amplification, Staining, Activation Assay, Binding Assay, Immunoprecipitation, Knock-Out

    24) Product Images from "Betulinic Acid Inhibits Cell Proliferation in Human Oral Squamous Cell Carcinoma via Modulating ROS-Regulated p53 Signaling"

    Article Title: Betulinic Acid Inhibits Cell Proliferation in Human Oral Squamous Cell Carcinoma via Modulating ROS-Regulated p53 Signaling

    Journal: Oncology Research

    doi: 10.3727/096504017X14841698396784

    Role of p53 in the antitumor effect of BA in vitro. KB cells were incubated with the indicated concentrations of BA for 24 h. (A, B) mRNA and protein expression of p53 were determined by real-time PCR and Western blot, respectively. (C) p53 binding in the promoter of Bax was examined by chromatin immunoprecipitation (ChIP) assay, and results are shown as folds of Bax promoter expression in the control group. (D) Reporter gene activity of p53 was examined and shown as relative activity of the control group. KB cells were transfected with LV-shp53 to knock down p53 expression. Cells in which p53 was stably knocked down were exposed to 100 μM BA for 24 h. (E) mRNA expression of p53 was measured to evaluate the efficiency of p53 knockdown. Apoptosis was examined by TUNEL assay (F), cell cycle distribution was measured by PI staining (G), and cell proliferation was determined by the CCK-8 kit (H). KB cells were exposed to 100 μM BA in the presence or absence of 100 μM NAC for 24 h. mRNA and protein expression of p53 were determined by real-time PCR and Western blot (I, J), respectively. * p
    Figure Legend Snippet: Role of p53 in the antitumor effect of BA in vitro. KB cells were incubated with the indicated concentrations of BA for 24 h. (A, B) mRNA and protein expression of p53 were determined by real-time PCR and Western blot, respectively. (C) p53 binding in the promoter of Bax was examined by chromatin immunoprecipitation (ChIP) assay, and results are shown as folds of Bax promoter expression in the control group. (D) Reporter gene activity of p53 was examined and shown as relative activity of the control group. KB cells were transfected with LV-shp53 to knock down p53 expression. Cells in which p53 was stably knocked down were exposed to 100 μM BA for 24 h. (E) mRNA expression of p53 was measured to evaluate the efficiency of p53 knockdown. Apoptosis was examined by TUNEL assay (F), cell cycle distribution was measured by PI staining (G), and cell proliferation was determined by the CCK-8 kit (H). KB cells were exposed to 100 μM BA in the presence or absence of 100 μM NAC for 24 h. mRNA and protein expression of p53 were determined by real-time PCR and Western blot (I, J), respectively. * p

    Techniques Used: In Vitro, Incubation, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Binding Assay, Chromatin Immunoprecipitation, Activity Assay, Transfection, Stable Transfection, TUNEL Assay, Staining, CCK-8 Assay

    Antitumor effect of BA in vivo. Mice (15 in each group) were implanted with tumors and treated with 50–150 mg/kg BA for 3 weeks. (A) Tumor volumes were calculated. (B) mRNA of p53 in tumors were determined by real-time PCR. (C) Phosphorylation of STAT3 in tumors was determined by Western blot. * p
    Figure Legend Snippet: Antitumor effect of BA in vivo. Mice (15 in each group) were implanted with tumors and treated with 50–150 mg/kg BA for 3 weeks. (A) Tumor volumes were calculated. (B) mRNA of p53 in tumors were determined by real-time PCR. (C) Phosphorylation of STAT3 in tumors was determined by Western blot. * p

    Techniques Used: In Vivo, Mouse Assay, Real-time Polymerase Chain Reaction, Western Blot

    25) Product Images from "DNA replication stress in CHK1-depleted tumour cells triggers premature (S-phase) mitosis through inappropriate activation of Aurora kinase B"

    Article Title: DNA replication stress in CHK1-depleted tumour cells triggers premature (S-phase) mitosis through inappropriate activation of Aurora kinase B

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2014.231

    CDK1 phosphorylation is maintained in CHK1-depleted HCT116 cells during DNA replication stress. Western blot analysis of indicated S-phase regulatory proteins from extracts of HCT116 or HCT116 p53−/− cells pretreated with CHK1 or control siRNAs and exposed or not exposed to thymidine (2 mM, 24 h). β -Actin levels are presented as loading controls
    Figure Legend Snippet: CDK1 phosphorylation is maintained in CHK1-depleted HCT116 cells during DNA replication stress. Western blot analysis of indicated S-phase regulatory proteins from extracts of HCT116 or HCT116 p53−/− cells pretreated with CHK1 or control siRNAs and exposed or not exposed to thymidine (2 mM, 24 h). β -Actin levels are presented as loading controls

    Techniques Used: Western Blot

    Ser10-histone H3 and Aurora kinase B phosphorylation in CHK1-depleted HCT116 p53−/− cells during replication stress are sensitive to Aurora B inhibitors. Western blot analysis of indicated S-phase regulatory and mitotic proteins from extracts of HCT116 p53−/− cells pretreated or not with CHK1 or control siRNAs and exposed or not exposed to thymidine (2 mM, 24 h). Effects of co-treatment with the Aurora A inhibitor MLN8237 (10 nM) or the Aurora B inhibitors AZD1152 (50 nM) and ZM447439 (2 μ M) are also presented. Inhibitors were added 1 h before cell collection. β -Actin levels are presented as loading controls
    Figure Legend Snippet: Ser10-histone H3 and Aurora kinase B phosphorylation in CHK1-depleted HCT116 p53−/− cells during replication stress are sensitive to Aurora B inhibitors. Western blot analysis of indicated S-phase regulatory and mitotic proteins from extracts of HCT116 p53−/− cells pretreated or not with CHK1 or control siRNAs and exposed or not exposed to thymidine (2 mM, 24 h). Effects of co-treatment with the Aurora A inhibitor MLN8237 (10 nM) or the Aurora B inhibitors AZD1152 (50 nM) and ZM447439 (2 μ M) are also presented. Inhibitors were added 1 h before cell collection. β -Actin levels are presented as loading controls

    Techniques Used: Western Blot

    Induction of aberrant mitoses and RPA foci in CHK1-depleted HCT116 and HCT116 p53−/− cells treated with thymidine. ( a ) Representative images of HCT116 p53−/− cells grown on coverslips were treated with CHK1 or control siRNAs in the presence or absence of thymidine (2 mM) for 24 h before fixation and staining for DAPI, pSer10 histone H3 and RPA2. These cells were then imaged by fluorescence microscopy. A typical mitosis in cells treated with control siRNA in the absence of thymidine is indicated by the white arrow in the top panel. Aberrant mitoses in CHK1 depleted cells treated with thymidine are indicated by yellow arrows. Several of the aberrant mitoses show clear RPA foci (red). ( b ) Representative multinucleate cells from the above cultures. Mean values for aberrant mitoses ( c ) or multinucleate cells ( d ) determined in three independent experiments ± S.D. values
    Figure Legend Snippet: Induction of aberrant mitoses and RPA foci in CHK1-depleted HCT116 and HCT116 p53−/− cells treated with thymidine. ( a ) Representative images of HCT116 p53−/− cells grown on coverslips were treated with CHK1 or control siRNAs in the presence or absence of thymidine (2 mM) for 24 h before fixation and staining for DAPI, pSer10 histone H3 and RPA2. These cells were then imaged by fluorescence microscopy. A typical mitosis in cells treated with control siRNA in the absence of thymidine is indicated by the white arrow in the top panel. Aberrant mitoses in CHK1 depleted cells treated with thymidine are indicated by yellow arrows. Several of the aberrant mitoses show clear RPA foci (red). ( b ) Representative multinucleate cells from the above cultures. Mean values for aberrant mitoses ( c ) or multinucleate cells ( d ) determined in three independent experiments ± S.D. values

    Techniques Used: Recombinase Polymerase Amplification, Staining, Fluorescence, Microscopy

    CDC45 depletion does not affect induction of pSer10-histone H3 in S-phase HCT116 or HCT116 p53−/− cells depleted of CHK1. ( a ) Indicated cells pretreated with CHK1 or CHK1 and CDC45 siRNAs were exposed or not exposed to 2 mM thymidine (TdR) for 24 h before collection and analysis for pSer10-histone H3 (top panels, scatter plots) and DNA content (bottom panels) by flow cytometry. ( b ) Western blot analysis of CDC45 and CHK1 depletion in a representative experiment. β -Actin levels are presented as loading controls. ( c ) Mean values (± S.D.) for three independent experiments as presented above
    Figure Legend Snippet: CDC45 depletion does not affect induction of pSer10-histone H3 in S-phase HCT116 or HCT116 p53−/− cells depleted of CHK1. ( a ) Indicated cells pretreated with CHK1 or CHK1 and CDC45 siRNAs were exposed or not exposed to 2 mM thymidine (TdR) for 24 h before collection and analysis for pSer10-histone H3 (top panels, scatter plots) and DNA content (bottom panels) by flow cytometry. ( b ) Western blot analysis of CDC45 and CHK1 depletion in a representative experiment. β -Actin levels are presented as loading controls. ( c ) Mean values (± S.D.) for three independent experiments as presented above

    Techniques Used: Flow Cytometry, Cytometry, Western Blot

    Induction of Ser10-histone H3 phosphorylation in S-phase CHK1-depleted HCT116 cells during DNA replication stress. ( a ) HCT116 cells pretreated with control or CHK1 siRNAs were exposed or not exposed to 2 mM thymidine (TdR) for 24 h before collection and analysis for pSer10 histone H3 (top panels, scatter plots) and DNA (bottom panels) content by flow cytometry. Cells with elevated pSer10-histone H3 are in the top quadrants of the scatter plots. S-phase cells showing phosphorylated histone H3 are in the top left quadrant those in G2/M are in the top right. Percentages of cells in each of the gated populations are indicated. ( b ) Mean values (± S.D.) for three independent experiments as presented above. The effects of nocodazole are also presented. ( c ) p53−/− HCT116 cells were treated with the CHK1 siRNA in the presence or absence of 2 mM thymidne for 24 h before collection and analysis of pSer10 Histone H3 and DNA content by flow cytometry. ( d ) Effects of nocodazole treatment on HCT116 or HCT116 p53−/− cells treated as in a , c
    Figure Legend Snippet: Induction of Ser10-histone H3 phosphorylation in S-phase CHK1-depleted HCT116 cells during DNA replication stress. ( a ) HCT116 cells pretreated with control or CHK1 siRNAs were exposed or not exposed to 2 mM thymidine (TdR) for 24 h before collection and analysis for pSer10 histone H3 (top panels, scatter plots) and DNA (bottom panels) content by flow cytometry. Cells with elevated pSer10-histone H3 are in the top quadrants of the scatter plots. S-phase cells showing phosphorylated histone H3 are in the top left quadrant those in G2/M are in the top right. Percentages of cells in each of the gated populations are indicated. ( b ) Mean values (± S.D.) for three independent experiments as presented above. The effects of nocodazole are also presented. ( c ) p53−/− HCT116 cells were treated with the CHK1 siRNA in the presence or absence of 2 mM thymidne for 24 h before collection and analysis of pSer10 Histone H3 and DNA content by flow cytometry. ( d ) Effects of nocodazole treatment on HCT116 or HCT116 p53−/− cells treated as in a , c

    Techniques Used: Flow Cytometry, Cytometry

    Aurora kinase autophosphorylation is required for Ser10-histone H3 phosphorylation in S-phase CHK1-depleted p53−/−HCT116 cells but is not required for cell death. ( a ) Control or CHK1-depleted HCT116 p53−/− cells were exposed or not exposed to 2 mM thymidine (TdR) for 24 h before collection and analysis of pSer10-histone H3, phospho-Aurora kinase and DNA content by flow cytometry. Cells showing elevated levels of the indicated phospho-proteins are gated and the % of cells presenting elevated levels is indicated. Effects of the Aurora B inhibitor (ZM447439, 2 μ M, 24 h) treatment are also presented. It is important to note that Aurora B inhibition has been reported to induce polyploidy; 34 however, when the cells are treated with ZM447439 and thymidine, they fail to traverse mitosis. ( b ) Mean values (± S.D.) for three independent experiments as presented above. ( c ) CHK1-depleted HCT116 p53−/− cells exposed to thymidine for 24 h were fixed and stained for pSer10-histone H3 and phospho-Aurora kinase content using mouse anti-pSer10-histone H3 and rabbit anti-phospho Aurora kinase. Alexa Fluor 647—R-Phycoerythrin goat anti-mouse IgG and FITC goat anti-rabbit IgG were then used to visualize the proteins by flow cytometry. Cells gated for phospho-Aurora kinase were then analysed for pSer10-histone H3. Similarly, cells gated for pSer10-histone H3 were subsequently analysed for phospho-Aurora kinase. Analyses show that gated cell populations co-stain for both proteins. ( d ) CHK1-depleted HCT116 p53−/− cells exposed to 2 mM thymidine for the indicated times in the presence or absence of 2 μ M ZM447439 were collected and analysed for DNA and pSer10-histone H3 content by flow cytometry. The percentages of cells with a subG1 DNA content or showing histone H3 phosphorylation presented are mean values from three independent experiments ±S.D.
    Figure Legend Snippet: Aurora kinase autophosphorylation is required for Ser10-histone H3 phosphorylation in S-phase CHK1-depleted p53−/−HCT116 cells but is not required for cell death. ( a ) Control or CHK1-depleted HCT116 p53−/− cells were exposed or not exposed to 2 mM thymidine (TdR) for 24 h before collection and analysis of pSer10-histone H3, phospho-Aurora kinase and DNA content by flow cytometry. Cells showing elevated levels of the indicated phospho-proteins are gated and the % of cells presenting elevated levels is indicated. Effects of the Aurora B inhibitor (ZM447439, 2 μ M, 24 h) treatment are also presented. It is important to note that Aurora B inhibition has been reported to induce polyploidy; 34 however, when the cells are treated with ZM447439 and thymidine, they fail to traverse mitosis. ( b ) Mean values (± S.D.) for three independent experiments as presented above. ( c ) CHK1-depleted HCT116 p53−/− cells exposed to thymidine for 24 h were fixed and stained for pSer10-histone H3 and phospho-Aurora kinase content using mouse anti-pSer10-histone H3 and rabbit anti-phospho Aurora kinase. Alexa Fluor 647—R-Phycoerythrin goat anti-mouse IgG and FITC goat anti-rabbit IgG were then used to visualize the proteins by flow cytometry. Cells gated for phospho-Aurora kinase were then analysed for pSer10-histone H3. Similarly, cells gated for pSer10-histone H3 were subsequently analysed for phospho-Aurora kinase. Analyses show that gated cell populations co-stain for both proteins. ( d ) CHK1-depleted HCT116 p53−/− cells exposed to 2 mM thymidine for the indicated times in the presence or absence of 2 μ M ZM447439 were collected and analysed for DNA and pSer10-histone H3 content by flow cytometry. The percentages of cells with a subG1 DNA content or showing histone H3 phosphorylation presented are mean values from three independent experiments ±S.D.

    Techniques Used: Flow Cytometry, Cytometry, Inhibition, Staining

    26) Product Images from "Evidence That Selenium Binding Protein 1 Is a Tumor Suppressor in Prostate Cancer"

    Article Title: Evidence That Selenium Binding Protein 1 Is a Tumor Suppressor in Prostate Cancer

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0127295

    SBP1 induction results in an increase in phospho-p53 and a decrease in total p53 in HCT116 cells. Total cell extracts from doxycycline treated or non-treated HCT116-TetSBP1 cells were analyzed using immunobloting for changes in phosphorylated Ser15 on p53 (A) and total p53 (B) levels in response to induction of SBP1 using anti-human SBP1, phospho p53-Ser15, p53, and β-Actin antibodies. β-Actin was used as an endogenous control.
    Figure Legend Snippet: SBP1 induction results in an increase in phospho-p53 and a decrease in total p53 in HCT116 cells. Total cell extracts from doxycycline treated or non-treated HCT116-TetSBP1 cells were analyzed using immunobloting for changes in phosphorylated Ser15 on p53 (A) and total p53 (B) levels in response to induction of SBP1 using anti-human SBP1, phospho p53-Ser15, p53, and β-Actin antibodies. β-Actin was used as an endogenous control.

    Techniques Used: Western Blot

    27) Product Images from "Melatonin Treatment Improves Mesenchymal Stem Cells Therapy by Preserving Stemness during Long-term In Vitro Expansion"

    Article Title: Melatonin Treatment Improves Mesenchymal Stem Cells Therapy by Preserving Stemness during Long-term In Vitro Expansion

    Journal: Theranostics

    doi: 10.7150/thno.15412

    Melatonin treatment decreases ROS levels, inhibits p53 pathway, and preserves NANOG expression in long-term passaged BMMSCs. (A-F) In vitro passaged rat BMMSCs of P1, P4, P15 treated with DMSO or 10 nM melatonin were used in following analysis. (A) ROS levels of BMMSCs were measured by flow cytometer (n=3). (B) Sod2 mRNA levels of BMMSCs were detected by real-time RT-PCR. β-actin was used as the loading control for quantification (n=3). (C) Expression of SOD2 protein was detected by western blot analysis. β-actin was used as the loading control (n=3). (D and E) Real-time RT-PCR was performed to measure p53 , p16 (D) and Nanog (E) mRNA levels in BMMSCs (n=3). (F) Expression of p53, p16 and NANOG protein was detected by western blotting (n=3). (G-P) BMMSCs were treated with DMSO, melatonin or melatonin plus LUZ from the 1 st passage to the 15 th passage. (G) Mel-1A-R and Mel-1B-R mRNA levels in BMMSCs were analysis by realtime RT-PCR. (n=3). (H) CFU-F formation of BMMSCs was detected by toluidine blue staining and calculated (n=3). (I) Proliferation of BMMSCs was detected from day 0 to day 6 (n=3). (J) ALP staining and ELISA analysis were performed to detect the activity of ALP of BMMSCs after 7-day osteogenic induction (n=3). (K) Mineralized nodules formed by BMMSCs were tested by alizarin red staining after 28-day osteogenic induction and quantified (n=3). (L-N) Runx2 , Ocn (L) , p53 , p16 (M) and Nanog (N) mRNA levels in BMMSCs were analyzed by Realtime RT-PCR (n=3). (O) ROS levels and Sod2 mRNA levels in BMMSCs (n=3). (P) Expression of Mel-1A-R, Mel-1B-R, SOD2, p53, p16 and NANOG protein was detected by western blot analysis. (n=3). Data are shown as mean±SD. P value is presented in each graph.
    Figure Legend Snippet: Melatonin treatment decreases ROS levels, inhibits p53 pathway, and preserves NANOG expression in long-term passaged BMMSCs. (A-F) In vitro passaged rat BMMSCs of P1, P4, P15 treated with DMSO or 10 nM melatonin were used in following analysis. (A) ROS levels of BMMSCs were measured by flow cytometer (n=3). (B) Sod2 mRNA levels of BMMSCs were detected by real-time RT-PCR. β-actin was used as the loading control for quantification (n=3). (C) Expression of SOD2 protein was detected by western blot analysis. β-actin was used as the loading control (n=3). (D and E) Real-time RT-PCR was performed to measure p53 , p16 (D) and Nanog (E) mRNA levels in BMMSCs (n=3). (F) Expression of p53, p16 and NANOG protein was detected by western blotting (n=3). (G-P) BMMSCs were treated with DMSO, melatonin or melatonin plus LUZ from the 1 st passage to the 15 th passage. (G) Mel-1A-R and Mel-1B-R mRNA levels in BMMSCs were analysis by realtime RT-PCR. (n=3). (H) CFU-F formation of BMMSCs was detected by toluidine blue staining and calculated (n=3). (I) Proliferation of BMMSCs was detected from day 0 to day 6 (n=3). (J) ALP staining and ELISA analysis were performed to detect the activity of ALP of BMMSCs after 7-day osteogenic induction (n=3). (K) Mineralized nodules formed by BMMSCs were tested by alizarin red staining after 28-day osteogenic induction and quantified (n=3). (L-N) Runx2 , Ocn (L) , p53 , p16 (M) and Nanog (N) mRNA levels in BMMSCs were analyzed by Realtime RT-PCR (n=3). (O) ROS levels and Sod2 mRNA levels in BMMSCs (n=3). (P) Expression of Mel-1A-R, Mel-1B-R, SOD2, p53, p16 and NANOG protein was detected by western blot analysis. (n=3). Data are shown as mean±SD. P value is presented in each graph.

    Techniques Used: Expressing, In Vitro, Flow Cytometry, Cytometry, Quantitative RT-PCR, Western Blot, Reverse Transcription Polymerase Chain Reaction, Staining, ALP Assay, Enzyme-linked Immunosorbent Assay, Activity Assay

    28) Product Images from "An improved auxin-inducible degron system preserves native protein levels and enables rapid and specific protein depletion"

    Article Title: An improved auxin-inducible degron system preserves native protein levels and enables rapid and specific protein depletion

    Journal: Genes & Development

    doi: 10.1101/gad.328237.119

    Endogenous tagging of genes with AID results in auxin-independent protein depletion. ( A ) targeting the 3′ end of the coding sequence with a homology-directed repair donor construct. The donor construct contained the AID domain fused to a hygromycin resistance marker; an intervening porcine teschovirus-1 2A (P2A) site liberates the hygromycin resistance marker during protein translation. This construct was flanked by 50-base-pair (bp) homology regions that correspond to the sequences flanking the sgRNA recognition sequence and Cas9 cleavage site. ( B – D ) We quantified AID-tagged protein abundance with quantitative Western blots and a dilution of the progenitor cell line for the standard curve. Endogenous homozygous tagging of ZNF143 ( B ), TEAD4 ( C ), and p53 ( D ) in HEK293T cells results in auxin-independent chronic protein depletion. MCF-7 ( E ) and DLD-1 ( F ) cell lines also exhibit depletion of endogenously tagged ZNF143 ( E ) and CENP-I ( F ).
    Figure Legend Snippet: Endogenous tagging of genes with AID results in auxin-independent protein depletion. ( A ) targeting the 3′ end of the coding sequence with a homology-directed repair donor construct. The donor construct contained the AID domain fused to a hygromycin resistance marker; an intervening porcine teschovirus-1 2A (P2A) site liberates the hygromycin resistance marker during protein translation. This construct was flanked by 50-base-pair (bp) homology regions that correspond to the sequences flanking the sgRNA recognition sequence and Cas9 cleavage site. ( B – D ) We quantified AID-tagged protein abundance with quantitative Western blots and a dilution of the progenitor cell line for the standard curve. Endogenous homozygous tagging of ZNF143 ( B ), TEAD4 ( C ), and p53 ( D ) in HEK293T cells results in auxin-independent chronic protein depletion. MCF-7 ( E ) and DLD-1 ( F ) cell lines also exhibit depletion of endogenously tagged ZNF143 ( E ) and CENP-I ( F ).

    Techniques Used: Sequencing, Construct, Marker, Western Blot

    29) Product Images from "The high‐risk HPV E6 proteins modify the activity of the eIF4E protein via the MEK/ERK and AKT/PKB pathways"

    Article Title: The high‐risk HPV E6 proteins modify the activity of the eIF4E protein via the MEK/ERK and AKT/PKB pathways

    Journal: FEBS Open Bio

    doi: 10.1002/2211-5463.12987

    Oncogenic E6 proteins increase eIF4E phosphorylation (Serine‐209). (A) Steady‐state levels of p53, and the total and phosphorylated (Ser‐209) forms of the eIF4E protein were evaluated via immunoblot assays in HEK293 (p53: WT), MCF7 (p53: WT), and HaCat (p53: mutated) cells. Band intensity was quantified by the densitometric analysis using imagej software. At least three experiments were performed for analysis. Differences were compared by one‐way ANOVA. (B) Expression level of eIF4E mRNA in stable cells transfected with different E6 constructs (HPV‐6 E6, HPV‐16 E6, HPV‐18 E6, and HPV‐52 E6) was evaluated by real‐time RT–PCR. Control cells were transfected with empty vector (vector). Values represent mean ± SD of at least three experiments. Differences between groups were compared by one‐way ANOVA with Dunnett’s post hoc test (* P > 0.05; ns: no significance). (C) Total lysates of HEK293, HaCat, and MCF7 cells transfected with the different E6 constructs were resolved by SDS/PAGE and immunoblotted with antibodies against p53, total and phosphorylated forms of eIF4E proteins. GAPDH was employed as a loading control for RT–PCR and western blot analysis. D, Relationship between total and phosphorylated levels of the eIF4E protein in transfected cells was evaluated by densitometric analysis. At least three experiments were performed for the evaluation.
    Figure Legend Snippet: Oncogenic E6 proteins increase eIF4E phosphorylation (Serine‐209). (A) Steady‐state levels of p53, and the total and phosphorylated (Ser‐209) forms of the eIF4E protein were evaluated via immunoblot assays in HEK293 (p53: WT), MCF7 (p53: WT), and HaCat (p53: mutated) cells. Band intensity was quantified by the densitometric analysis using imagej software. At least three experiments were performed for analysis. Differences were compared by one‐way ANOVA. (B) Expression level of eIF4E mRNA in stable cells transfected with different E6 constructs (HPV‐6 E6, HPV‐16 E6, HPV‐18 E6, and HPV‐52 E6) was evaluated by real‐time RT–PCR. Control cells were transfected with empty vector (vector). Values represent mean ± SD of at least three experiments. Differences between groups were compared by one‐way ANOVA with Dunnett’s post hoc test (* P > 0.05; ns: no significance). (C) Total lysates of HEK293, HaCat, and MCF7 cells transfected with the different E6 constructs were resolved by SDS/PAGE and immunoblotted with antibodies against p53, total and phosphorylated forms of eIF4E proteins. GAPDH was employed as a loading control for RT–PCR and western blot analysis. D, Relationship between total and phosphorylated levels of the eIF4E protein in transfected cells was evaluated by densitometric analysis. At least three experiments were performed for the evaluation.

    Techniques Used: Software, Expressing, Transfection, Construct, Quantitative RT-PCR, Plasmid Preparation, SDS Page, Reverse Transcription Polymerase Chain Reaction, Western Blot

    30) Product Images from "Co-culture with lung cancer A549 cells promotes the proliferation and migration of mesenchymal stem cells derived from bone marrow"

    Article Title: Co-culture with lung cancer A549 cells promotes the proliferation and migration of mesenchymal stem cells derived from bone marrow

    Journal: Experimental and Therapeutic Medicine

    doi: 10.3892/etm.2017.4909

    Western blot analysis of the expression of proteins involved in proliferation and differentiation in the different cell groups. (A) Western blotting identified the differential protein expression of Bax, Bcl-2, caspase-3, P53, P62 (c-Myc), Ras, P-ERK and NF-κB in BM-MSCs, co-BM-MSCs and A549 cells. β-actin was used as a loading control. (B) Relative levels of Bcl-2, Bax, caspase-3, HDAC4, P53, P62 (c-Myc), Ras, P-ERK and NF-κB expression in BM-MSCs, co-BM-MSCs and A549 cells. Compared with the BM-MSC group, levels of Ras, P-ERK, NF-κB, P62 and Bcl-2 expression in the co-BM-MSCs group were significantly increased and the levels of P53, Bax and caspase-3 expression decreased significantly. Data are presented as the mean ± standard deviation of three individual experiments and calculated as relative levels of controls. *P
    Figure Legend Snippet: Western blot analysis of the expression of proteins involved in proliferation and differentiation in the different cell groups. (A) Western blotting identified the differential protein expression of Bax, Bcl-2, caspase-3, P53, P62 (c-Myc), Ras, P-ERK and NF-κB in BM-MSCs, co-BM-MSCs and A549 cells. β-actin was used as a loading control. (B) Relative levels of Bcl-2, Bax, caspase-3, HDAC4, P53, P62 (c-Myc), Ras, P-ERK and NF-κB expression in BM-MSCs, co-BM-MSCs and A549 cells. Compared with the BM-MSC group, levels of Ras, P-ERK, NF-κB, P62 and Bcl-2 expression in the co-BM-MSCs group were significantly increased and the levels of P53, Bax and caspase-3 expression decreased significantly. Data are presented as the mean ± standard deviation of three individual experiments and calculated as relative levels of controls. *P

    Techniques Used: Western Blot, Expressing, Standard Deviation

    31) Product Images from "Anti-inflammatory activity of chloroquine and amodiaquine through p21-mediated suppression of T cell proliferation and Th1 cell differentiation"

    Article Title: Anti-inflammatory activity of chloroquine and amodiaquine through p21-mediated suppression of T cell proliferation and Th1 cell differentiation

    Journal: Biochemical and biophysical research communications

    doi: 10.1016/j.bbrc.2016.04.105

    Induction of p21 protein expression by CQ and more prominently by AQ. (A–B) TCR-triggered CD4+ T cells were incubated with different amounts of CQ or AQ for 24 h. Whole cell extracts were resolved by SDS-PAGE and subjected to immunoblotting with antibody against p21, p27, PCNA, p53, LC3B, ATG5, or actin (A). The intensity of the p21 protein band was quantitatively analyzed by a densitometry scan (B). (C) CQ- or AQ-treated T cells were fixed in 4% paraformaldehyde and stained with antibody against p21 and LAMP1, followed by subsequent incubation with Alexa Fluor 594- and Alexa Fluor 488-conjugated secondary antibody. The nuclei were blue counterstained with DAPI. (D) Activated CD4+ T cells were treated with cycloheximide (+CHX) in the presence of either CQ or AQ and harvested for immunoblotting analysis. (E) Total RNA was obtained from CQ- or AQ-treated T cells and subjected to reverse transcription and real time-PCR analysis. The relative level of p21 mRNA was calculated after normalization to the actin level. *, p
    Figure Legend Snippet: Induction of p21 protein expression by CQ and more prominently by AQ. (A–B) TCR-triggered CD4+ T cells were incubated with different amounts of CQ or AQ for 24 h. Whole cell extracts were resolved by SDS-PAGE and subjected to immunoblotting with antibody against p21, p27, PCNA, p53, LC3B, ATG5, or actin (A). The intensity of the p21 protein band was quantitatively analyzed by a densitometry scan (B). (C) CQ- or AQ-treated T cells were fixed in 4% paraformaldehyde and stained with antibody against p21 and LAMP1, followed by subsequent incubation with Alexa Fluor 594- and Alexa Fluor 488-conjugated secondary antibody. The nuclei were blue counterstained with DAPI. (D) Activated CD4+ T cells were treated with cycloheximide (+CHX) in the presence of either CQ or AQ and harvested for immunoblotting analysis. (E) Total RNA was obtained from CQ- or AQ-treated T cells and subjected to reverse transcription and real time-PCR analysis. The relative level of p21 mRNA was calculated after normalization to the actin level. *, p

    Techniques Used: Expressing, Incubation, SDS Page, Staining, Real-time Polymerase Chain Reaction

    32) Product Images from "Mucinous adenocarcinoma developed from human fallopian tube epithelial cells through defined genetic modifications"

    Article Title: Mucinous adenocarcinoma developed from human fallopian tube epithelial cells through defined genetic modifications

    Journal: Cell Cycle

    doi: 10.4161/cc.20544

    Figure 2. Immunohistological characteristics of HRAS V12 -transformed FTEs and tumor xenografts. Immunohistochemical staining to cytokeratin, p53, SV40, WT-1, PAX8 and PAX2 in FTE187-hT-SV-H cells (A–C, G–I) as well as in tumor xenographs
    Figure Legend Snippet: Figure 2. Immunohistological characteristics of HRAS V12 -transformed FTEs and tumor xenografts. Immunohistochemical staining to cytokeratin, p53, SV40, WT-1, PAX8 and PAX2 in FTE187-hT-SV-H cells (A–C, G–I) as well as in tumor xenographs

    Techniques Used: Transformation Assay, Immunohistochemistry, Staining

    33) Product Images from "A gain-of-function mutant p53-HSF1 feed forward circuit governs adaptation of cancer cells to proteotoxic stress"

    Article Title: A gain-of-function mutant p53-HSF1 feed forward circuit governs adaptation of cancer cells to proteotoxic stress

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2014.158

    Mutant p53 protein enhances binding of HSF1 to HSE elements and stimulates its transcriptional activity. ( a ) Tet-inducible mutp53 knockdown inhibits the heat-shock response at the transcriptional level. Hsp70 mRNA levels measured by qRT-PCR± HS. Two independent experiments were performed in triplicate. * P
    Figure Legend Snippet: Mutant p53 protein enhances binding of HSF1 to HSE elements and stimulates its transcriptional activity. ( a ) Tet-inducible mutp53 knockdown inhibits the heat-shock response at the transcriptional level. Hsp70 mRNA levels measured by qRT-PCR± HS. Two independent experiments were performed in triplicate. * P

    Techniques Used: Mutagenesis, Binding Assay, Activity Assay, Quantitative RT-PCR

    HSF1 activation by mutant p53 renders cells resistant to proteotoxic stress. ( a ) The elevated levels of mutp53 in MDA231R280K cells bestow increased thermotolerance compared with parental controls. Cells were grown at 37 °C or heat-shocked for 1 h at 43 °C. After 48 h, viability was measured using CTB assay. Two independent experiments were performed in triplicate. ( b ) Conversely, downregulation of mutp53 decreases thermotolerance. Scrambled or p53 siRNA was transfected into MDA231 cells. After 24-h post transfection, cells were heat-shocked for 1 h at 43 °C. Left, CTB assay after 48 h. Right, corresponding immunoblot. ( c ) Primary MECs from p53H/H;ErbB2 mice acquire higher tolerance to proteotoxic stress from HS (1 h, 43 °C) or proteasome inhibition (MG132, 1 μ M for 24 h) and exhibit higher levels of HSF1 and its targets Hsp70 and Hsp27, compared with p53−/−ErbB2 MECs. Two independent experiments were performed in triplicate. ( d ) The elevated levels of mutp53 in MDA231R280K cells promote their chemoresistance to genotoxic Camptothecin (50 nM, 48 h). Viability assay and PARP cleavage. MAPK, loading control. ( a – d ) Error bars represent mean±S.D.
    Figure Legend Snippet: HSF1 activation by mutant p53 renders cells resistant to proteotoxic stress. ( a ) The elevated levels of mutp53 in MDA231R280K cells bestow increased thermotolerance compared with parental controls. Cells were grown at 37 °C or heat-shocked for 1 h at 43 °C. After 48 h, viability was measured using CTB assay. Two independent experiments were performed in triplicate. ( b ) Conversely, downregulation of mutp53 decreases thermotolerance. Scrambled or p53 siRNA was transfected into MDA231 cells. After 24-h post transfection, cells were heat-shocked for 1 h at 43 °C. Left, CTB assay after 48 h. Right, corresponding immunoblot. ( c ) Primary MECs from p53H/H;ErbB2 mice acquire higher tolerance to proteotoxic stress from HS (1 h, 43 °C) or proteasome inhibition (MG132, 1 μ M for 24 h) and exhibit higher levels of HSF1 and its targets Hsp70 and Hsp27, compared with p53−/−ErbB2 MECs. Two independent experiments were performed in triplicate. ( d ) The elevated levels of mutp53 in MDA231R280K cells promote their chemoresistance to genotoxic Camptothecin (50 nM, 48 h). Viability assay and PARP cleavage. MAPK, loading control. ( a – d ) Error bars represent mean±S.D.

    Techniques Used: Activation Assay, Mutagenesis, CtB Assay, Transfection, Mouse Assay, Inhibition, Viability Assay

    Mutant p53 promotes HSF1 activation via Ser326 phosphorylation. ( a ) Activated p-Ser326 HSF1 is greatly upregulated in MDA231R280K cells after HS (43 °C, 1 h). Actin is the loading control. ( b ) MDA231R280K cells induce higher levels of activated nuclear p-Ser326 HSF1 after HS than vector controls. Note that the slower migration of HS-activated HSF1 detectable in 6% SDS PAAG is due to phosphorylation. ( c ) Tet-inducible p53 shRNA decreases total HSF1 and activated p-Ser326 HSF1 in the nuclear fraction of MDA231 cells after HS (43 °C, 1 h) compared with vehicle-treated control cells. Note that the slower migration of HS-activated HSF1 detectable in 6% SDS PAAG is due to phosphorylation. ( d ) HSF1 is specifically upregulated by mutant but not wild-type p53. No increase in HSF1 levels or activation was seen in HCT116 p53−/− versus p53+/+ cells. The slower migration of total HSF1 detectable in 6% SDS PAAG upon HS is due to phosphorylation. ( b – d ) N – nuclear fraction, C – cytoplasmic fraction. GAPDH and HDAC1 as cytoplasmic and nuclear markers, respectively
    Figure Legend Snippet: Mutant p53 promotes HSF1 activation via Ser326 phosphorylation. ( a ) Activated p-Ser326 HSF1 is greatly upregulated in MDA231R280K cells after HS (43 °C, 1 h). Actin is the loading control. ( b ) MDA231R280K cells induce higher levels of activated nuclear p-Ser326 HSF1 after HS than vector controls. Note that the slower migration of HS-activated HSF1 detectable in 6% SDS PAAG is due to phosphorylation. ( c ) Tet-inducible p53 shRNA decreases total HSF1 and activated p-Ser326 HSF1 in the nuclear fraction of MDA231 cells after HS (43 °C, 1 h) compared with vehicle-treated control cells. Note that the slower migration of HS-activated HSF1 detectable in 6% SDS PAAG is due to phosphorylation. ( d ) HSF1 is specifically upregulated by mutant but not wild-type p53. No increase in HSF1 levels or activation was seen in HCT116 p53−/− versus p53+/+ cells. The slower migration of total HSF1 detectable in 6% SDS PAAG upon HS is due to phosphorylation. ( b – d ) N – nuclear fraction, C – cytoplasmic fraction. GAPDH and HDAC1 as cytoplasmic and nuclear markers, respectively

    Techniques Used: Mutagenesis, Activation Assay, Plasmid Preparation, Migration, shRNA

    Upstream EGFR/ErbB2 signaling mediates HSF1 activation in a mutp53-dependent manner. ( a ) Dual inhibition of EGFR/ErbB2 inhibits HSF1 activation. ErbB2+ breast cancer cells SKBr3 were treated with CP724714 (10 μ M for 24 h) or vehicle. ( a – h ) Immunoblot analysis. ( b ) Stimulation of EGFR signaling induces Ser326 HSF1 phosphorylation. MDA231 cells serum-starved overnight and treated with EGF (30 ng/ml) for 10 min before harvesting. ( c ) Overexpression of mutp53 potentiates EGFR signaling after EGF stimulation. Increased phosphorylation of EGFR-Tyr845 and downstream effectors in MDA231R280K vs vector controls. Treatment as in b . ( d ) Stable overexpression of native p53R175H in SKBr3 increases the levels of ErbB2, pAKT and the HSF1 response. ( e ) Depletion of mutp53 or HSF1 in SKBr3 cells reduces both EGFR and p-Ser326 HSF1 levels. Transfection with scrambled, p53 or HSF1 siRNAs. ( f ) Left, the mutp53 allele in MECs from p53H/+ErbB2 mice correlates with increased levels of ErbB2 and higher activity of HSF1, compared with the p53 null allele. Right , even in the absence of the ErbB2 transgene, MECs from p53H/H mice have higher levels of ErbB2 and heat-shock proteins than p53−/− MECs. ( g ) Cell lines, established from primary mammary tumors of H/H;ErbB2 mouse showed elevated levels of ErbB2, EGFR and activated HSF1 (indicated by increased Hsp70) compared with p53−/−ErbB2 littermate control. ( h ) Dual inhibition of PI3K and MAPK signaling decreases EGFR levels in a mutp53-dependent manner. MDA231 vector and MDA231R280K cells were treated with low or high concentrations of UO126 (10 or 20 μ M) and LY294002 (5 or 10 μ M) for 48 h. ( i ) p53 and p-Ser326 HSF1 immunohistochemical staining of tissue microarray of 150 primary invasive human breast cancers from 75 different patients (two biopsies from separate tumor regions per patient). Representative cases are shown. Strong p53 staining was used as surrogate marker for p53 mutation and a nuclear signal with a p-Ser326-specific HSF1 antibody as marker for HSF1 activation. Staining intensities were blindly scored from 0 to 4. A clear correlation between levels of mutp53 stabilization and nuclear p-Ser326 HSF1 were present in strong (3+) Her2-positive tumors (rho=0.213, P =0.008; n =85 cores) but not in Her2 weak or negative tumors (rho=0.243, P =0.932; n =65 cores). See also Supplementary Figure 1 . ( j ) Proposed model. mutp53, by enhancing EGFR/ErbB2/MAPK/PI3K signaling, potentiates HSF1 activity via a feed forward circuit and thereby endows cancer cells with superior tolerance to proteotoxic stress
    Figure Legend Snippet: Upstream EGFR/ErbB2 signaling mediates HSF1 activation in a mutp53-dependent manner. ( a ) Dual inhibition of EGFR/ErbB2 inhibits HSF1 activation. ErbB2+ breast cancer cells SKBr3 were treated with CP724714 (10 μ M for 24 h) or vehicle. ( a – h ) Immunoblot analysis. ( b ) Stimulation of EGFR signaling induces Ser326 HSF1 phosphorylation. MDA231 cells serum-starved overnight and treated with EGF (30 ng/ml) for 10 min before harvesting. ( c ) Overexpression of mutp53 potentiates EGFR signaling after EGF stimulation. Increased phosphorylation of EGFR-Tyr845 and downstream effectors in MDA231R280K vs vector controls. Treatment as in b . ( d ) Stable overexpression of native p53R175H in SKBr3 increases the levels of ErbB2, pAKT and the HSF1 response. ( e ) Depletion of mutp53 or HSF1 in SKBr3 cells reduces both EGFR and p-Ser326 HSF1 levels. Transfection with scrambled, p53 or HSF1 siRNAs. ( f ) Left, the mutp53 allele in MECs from p53H/+ErbB2 mice correlates with increased levels of ErbB2 and higher activity of HSF1, compared with the p53 null allele. Right , even in the absence of the ErbB2 transgene, MECs from p53H/H mice have higher levels of ErbB2 and heat-shock proteins than p53−/− MECs. ( g ) Cell lines, established from primary mammary tumors of H/H;ErbB2 mouse showed elevated levels of ErbB2, EGFR and activated HSF1 (indicated by increased Hsp70) compared with p53−/−ErbB2 littermate control. ( h ) Dual inhibition of PI3K and MAPK signaling decreases EGFR levels in a mutp53-dependent manner. MDA231 vector and MDA231R280K cells were treated with low or high concentrations of UO126 (10 or 20 μ M) and LY294002 (5 or 10 μ M) for 48 h. ( i ) p53 and p-Ser326 HSF1 immunohistochemical staining of tissue microarray of 150 primary invasive human breast cancers from 75 different patients (two biopsies from separate tumor regions per patient). Representative cases are shown. Strong p53 staining was used as surrogate marker for p53 mutation and a nuclear signal with a p-Ser326-specific HSF1 antibody as marker for HSF1 activation. Staining intensities were blindly scored from 0 to 4. A clear correlation between levels of mutp53 stabilization and nuclear p-Ser326 HSF1 were present in strong (3+) Her2-positive tumors (rho=0.213, P =0.008; n =85 cores) but not in Her2 weak or negative tumors (rho=0.243, P =0.932; n =65 cores). See also Supplementary Figure 1 . ( j ) Proposed model. mutp53, by enhancing EGFR/ErbB2/MAPK/PI3K signaling, potentiates HSF1 activity via a feed forward circuit and thereby endows cancer cells with superior tolerance to proteotoxic stress

    Techniques Used: Activation Assay, Inhibition, Over Expression, Plasmid Preparation, Transfection, Mouse Assay, Activity Assay, Immunohistochemistry, Staining, Microarray, Marker, Mutagenesis

    Mutant p53 interacts with activated HSF1 in the nucleus. ( a ) mutp53-HSF1 complexes are induced by HS (43 °C, 1 h). Whole-cell lysates of MDA231R280K cells were immunoprecipitated with p53 or irrelevant GST antibodies and immunoblotted for HSF1 and p53. * – the slower migration band of total HSF1 detectable in 6% SDS PAAG corresponds to phosphorylated form of HSF1. ( b ) Likewise, the endogenous mutp53-HSF1 complex in the nucleus is induced by HS. Nuclear and cytoplasmic fractions from MDA231 cells±HS were immunoprecipitated with HSF1 or irrelevant GFP antibodies and blotted for p53 and HSF1. HSP90 and HDAC1 as cytoplasmic and nuclear markers, respectively. ( c ) Endogenous mutp53 specifically interacts with the activated p-Ser326 form of HSF1 after HS. Nuclear fractions of MDA231 cells were precipitated with antibodies to HSF1, p-Ser326 HSF1 or GFP. Immunoblot loading normalized for similar amounts of immunoprecipitated total HSF1. ( d ) HSF1 does not interact with wild-type p53. Cell lysates from heat-shocked HCT116 p53+/+ or MDA213 cells were immunoprecipitated for HSF1 or GFP and blotted for HSF1 and p53
    Figure Legend Snippet: Mutant p53 interacts with activated HSF1 in the nucleus. ( a ) mutp53-HSF1 complexes are induced by HS (43 °C, 1 h). Whole-cell lysates of MDA231R280K cells were immunoprecipitated with p53 or irrelevant GST antibodies and immunoblotted for HSF1 and p53. * – the slower migration band of total HSF1 detectable in 6% SDS PAAG corresponds to phosphorylated form of HSF1. ( b ) Likewise, the endogenous mutp53-HSF1 complex in the nucleus is induced by HS. Nuclear and cytoplasmic fractions from MDA231 cells±HS were immunoprecipitated with HSF1 or irrelevant GFP antibodies and blotted for p53 and HSF1. HSP90 and HDAC1 as cytoplasmic and nuclear markers, respectively. ( c ) Endogenous mutp53 specifically interacts with the activated p-Ser326 form of HSF1 after HS. Nuclear fractions of MDA231 cells were precipitated with antibodies to HSF1, p-Ser326 HSF1 or GFP. Immunoblot loading normalized for similar amounts of immunoprecipitated total HSF1. ( d ) HSF1 does not interact with wild-type p53. Cell lysates from heat-shocked HCT116 p53+/+ or MDA213 cells were immunoprecipitated for HSF1 or GFP and blotted for HSF1 and p53

    Techniques Used: Mutagenesis, Immunoprecipitation, Migration

    Mutant p53 upregulates HSF1 protein and augments the heat-shock response. ( a ) siRNA-mediated depletion of mutp53 in SKBr3 cells results in downregulation of HSF1 and its transcriptional target Hsp70. siHSF1 and scrambled siRNA as controls. ( a – c , e and f ) actin, loading control. ( b ) Tet-inducible p53 shRNA downregulates HSF1 and its target Hsp70 and is further enhanced by HS (43 °C, 1 h). Stable MDA231-shp53 cells. ( c ) Conversely, excess ectopic native mutp53 R280K in MDA231 cells further increases the HSF1 targets Hsp70/Hsp27 upon HS. ( d ) Ectopic mutp53R175H in p53 null H1299 cells upregulates HSF1 and its targets Hsp70/Hsp27, which is further enhanced by HS. Hsc70, loading control. ( e ) Upregulation of HSF1 and its targets in response to all tested p53 mutants. ( f ) Compared with littermate p53−/+ErbB2, primary mammary tumors from p53H/+ErbB2 mice show increased levels of HSF1 and Hsp70. Actin as a loading control
    Figure Legend Snippet: Mutant p53 upregulates HSF1 protein and augments the heat-shock response. ( a ) siRNA-mediated depletion of mutp53 in SKBr3 cells results in downregulation of HSF1 and its transcriptional target Hsp70. siHSF1 and scrambled siRNA as controls. ( a – c , e and f ) actin, loading control. ( b ) Tet-inducible p53 shRNA downregulates HSF1 and its target Hsp70 and is further enhanced by HS (43 °C, 1 h). Stable MDA231-shp53 cells. ( c ) Conversely, excess ectopic native mutp53 R280K in MDA231 cells further increases the HSF1 targets Hsp70/Hsp27 upon HS. ( d ) Ectopic mutp53R175H in p53 null H1299 cells upregulates HSF1 and its targets Hsp70/Hsp27, which is further enhanced by HS. Hsc70, loading control. ( e ) Upregulation of HSF1 and its targets in response to all tested p53 mutants. ( f ) Compared with littermate p53−/+ErbB2, primary mammary tumors from p53H/+ErbB2 mice show increased levels of HSF1 and Hsp70. Actin as a loading control

    Techniques Used: Mutagenesis, shRNA, Mouse Assay

    MAPK and PI3K cascades mediate HSF1 activation in a mutp53-dependent manner. ( a ) MEK 1/2 inhibition inhibits HSF1 activation in cancer cells. Immunoblot analysis of MDA231 cells treated overnight with 10 μ M U0126 or DMSO, followed by HS (43 °C for 1 h). ( b ) UO126 inhibition of HSF1 activation is more pronounced in mutp53-depleted cells. MDA231 stably expressing tet-inducible shp53 either vehicle- (−) or tetracycline (+)-treated, followed by 20 μ M U0126 for 24 h and ±HS (43 °C for 1 h). ( c ) Dual inhibition of the PI3K and MAPK pathways further impedes HSF1 activation after HS. Combined treatment of MDA231 with UO126 (20 μ M) and LY294002 (5 μ M) for 24 h. Inhibition is largely rescued by mutp53 overexpression in MDA231R280K cells. ( d ) As a result of HSF1 inhibition, PI3K (LY294002) and MAPK (UO126) inhibition significantly reduces the levels of Hsp90 α only in mutp53 MECs but only marginally in p53 null MECs from ErbB2 mice (10 μ M for 24 h each)
    Figure Legend Snippet: MAPK and PI3K cascades mediate HSF1 activation in a mutp53-dependent manner. ( a ) MEK 1/2 inhibition inhibits HSF1 activation in cancer cells. Immunoblot analysis of MDA231 cells treated overnight with 10 μ M U0126 or DMSO, followed by HS (43 °C for 1 h). ( b ) UO126 inhibition of HSF1 activation is more pronounced in mutp53-depleted cells. MDA231 stably expressing tet-inducible shp53 either vehicle- (−) or tetracycline (+)-treated, followed by 20 μ M U0126 for 24 h and ±HS (43 °C for 1 h). ( c ) Dual inhibition of the PI3K and MAPK pathways further impedes HSF1 activation after HS. Combined treatment of MDA231 with UO126 (20 μ M) and LY294002 (5 μ M) for 24 h. Inhibition is largely rescued by mutp53 overexpression in MDA231R280K cells. ( d ) As a result of HSF1 inhibition, PI3K (LY294002) and MAPK (UO126) inhibition significantly reduces the levels of Hsp90 α only in mutp53 MECs but only marginally in p53 null MECs from ErbB2 mice (10 μ M for 24 h each)

    Techniques Used: Activation Assay, Inhibition, Stable Transfection, Expressing, Over Expression, Mouse Assay

    34) Product Images from "p53-Dependent Senescence in Mesenchymal Stem Cells under Chronic Normoxia Is Potentiated by Low-Dose γ-Irradiation"

    Article Title: p53-Dependent Senescence in Mesenchymal Stem Cells under Chronic Normoxia Is Potentiated by Low-Dose γ-Irradiation

    Journal: Stem Cells International

    doi: 10.1155/2016/6429853

    Impact of p53 status on oxygen effect of long-term in vitro culturing and colony formation capacity of mMSCs. (a) Morphology of mMSCs p53−/− cultured in hypoxic conditions (left image) and in normoxic conditions (right image). Representative images show mMSCs p53−/− cultured for 20 days. (b) Colony forming ability of mMSCs p53−/− after 14 days in hypoxic (grey bar) and normoxic (black bar) cultures calculated as mean values of fibroblast colony-forming units (CFU-F) per plated cell (3 biological replicates).
    Figure Legend Snippet: Impact of p53 status on oxygen effect of long-term in vitro culturing and colony formation capacity of mMSCs. (a) Morphology of mMSCs p53−/− cultured in hypoxic conditions (left image) and in normoxic conditions (right image). Representative images show mMSCs p53−/− cultured for 20 days. (b) Colony forming ability of mMSCs p53−/− after 14 days in hypoxic (grey bar) and normoxic (black bar) cultures calculated as mean values of fibroblast colony-forming units (CFU-F) per plated cell (3 biological replicates).

    Techniques Used: In Vitro, Cell Culture

    Effects of oxygen levels on sensitivity of p53 wild-type mMSCs for radiation-induced senescence. (a) Green SA β -gal positive mMSCs in sham-irradiated (0 Gy) hypoxic (upper left) and in normoxic cultures (upper right) and 7 days after exposure to 4 Gy γ -irradiation (hypoxia, lower left, and normoxia, lower right). (b) Percentage of cells undergoing senescence in hypoxic and normoxic conditions after increasing doses of γ -irradiation depicted as mean values ± standard error of the mean (4 biological replicates), radiation dose on logarithmic scale.
    Figure Legend Snippet: Effects of oxygen levels on sensitivity of p53 wild-type mMSCs for radiation-induced senescence. (a) Green SA β -gal positive mMSCs in sham-irradiated (0 Gy) hypoxic (upper left) and in normoxic cultures (upper right) and 7 days after exposure to 4 Gy γ -irradiation (hypoxia, lower left, and normoxia, lower right). (b) Percentage of cells undergoing senescence in hypoxic and normoxic conditions after increasing doses of γ -irradiation depicted as mean values ± standard error of the mean (4 biological replicates), radiation dose on logarithmic scale.

    Techniques Used: Irradiation

    Effects of oxygen levels on long-term in vitro culturing and colony forming capacity of p53 wild-type mMSCs. (a) Proliferation and morphology of mMSCs p53wt/wt cultured in hypoxic conditions (2% O 2 , left) and in normoxic conditions (21% O 2 , right). Representative images show mMSCs cultured for 32 days. (b) Colony forming ability of mMSCs in hypoxic (upper) and normoxic (lower) atmospheres 14 days after plating 5,000 cells in a 75 cm 2 flask. (c) Plating efficiency calculated as mean values of fibroblast colony forming units (CFU-F) per plated cell ± standard error of the mean ( n = 4) in hypoxic (grey bar) and normoxic (black bar) conditions.
    Figure Legend Snippet: Effects of oxygen levels on long-term in vitro culturing and colony forming capacity of p53 wild-type mMSCs. (a) Proliferation and morphology of mMSCs p53wt/wt cultured in hypoxic conditions (2% O 2 , left) and in normoxic conditions (21% O 2 , right). Representative images show mMSCs cultured for 32 days. (b) Colony forming ability of mMSCs in hypoxic (upper) and normoxic (lower) atmospheres 14 days after plating 5,000 cells in a 75 cm 2 flask. (c) Plating efficiency calculated as mean values of fibroblast colony forming units (CFU-F) per plated cell ± standard error of the mean ( n = 4) in hypoxic (grey bar) and normoxic (black bar) conditions.

    Techniques Used: In Vitro, Cell Culture

    Impact of p53 status on senescence induction and number of DNA repair foci. (a) Graph shows percentage of p53−/− mMSCs undergoing senescence in hypoxic and normoxic conditions after exposure to γ -irradiation (sham-irradiated = 0 Gy, 3 biological replicates), radiation dose on logarithmic scale. (b) Quantification of p- γ H2AX foci in 50 p53−/− mMSC nuclei depicted as mean values ± standard error of the mean (3 biological replicates).
    Figure Legend Snippet: Impact of p53 status on senescence induction and number of DNA repair foci. (a) Graph shows percentage of p53−/− mMSCs undergoing senescence in hypoxic and normoxic conditions after exposure to γ -irradiation (sham-irradiated = 0 Gy, 3 biological replicates), radiation dose on logarithmic scale. (b) Quantification of p- γ H2AX foci in 50 p53−/− mMSC nuclei depicted as mean values ± standard error of the mean (3 biological replicates).

    Techniques Used: Irradiation

    35) Product Images from "Anticolorectal cancer effects and pharmacokinetic application of 2, 2-Bis [4-(4-amino-3-hydroxyphenoxy) phenyl] adamantane"

    Article Title: Anticolorectal cancer effects and pharmacokinetic application of 2, 2-Bis [4-(4-amino-3-hydroxyphenoxy) phenyl] adamantane

    Journal: International Journal of Clinical and Experimental Medicine

    doi:

    Effects of DPA on the growth of HCT-116, HCT-116 p53 -/- and HCT-116 p21 -/- cells. Each determination represents mean ± SD absorbance of three replicates. The data are representative of three reproducible independent experiments.
    Figure Legend Snippet: Effects of DPA on the growth of HCT-116, HCT-116 p53 -/- and HCT-116 p21 -/- cells. Each determination represents mean ± SD absorbance of three replicates. The data are representative of three reproducible independent experiments.

    Techniques Used:

    A. Immunoblot analysis of p-ERK, ERK, p21 and p53 in treated cells. HCT-116, HCT-116 p21 -/- and HCT-116 p53 -/- cells were treated with 0.1% DMSO (-) or 4 M DPA (+) for 48 hr. The Actin signal was used for normalization. B. The expression of E-cadherin,
    Figure Legend Snippet: A. Immunoblot analysis of p-ERK, ERK, p21 and p53 in treated cells. HCT-116, HCT-116 p21 -/- and HCT-116 p53 -/- cells were treated with 0.1% DMSO (-) or 4 M DPA (+) for 48 hr. The Actin signal was used for normalization. B. The expression of E-cadherin,

    Techniques Used: Expressing

    Effects of DPA on cell motility of HCT-116, HCT-116 p53 -/- and HCT-116 p21 -/- cells. HCT-116, HCT-116 p53 -/- and HCT-116 p21 -/- cells were treated with 0.1% DMSO or 4 M DPA for 48 hr, then seeded at a density of 1×10 5 cells/well in 24-well transwell
    Figure Legend Snippet: Effects of DPA on cell motility of HCT-116, HCT-116 p53 -/- and HCT-116 p21 -/- cells. HCT-116, HCT-116 p53 -/- and HCT-116 p21 -/- cells were treated with 0.1% DMSO or 4 M DPA for 48 hr, then seeded at a density of 1×10 5 cells/well in 24-well transwell

    Techniques Used:

    Effects of DPA on cell cycle progression of HCT-116, HCT-116 p53 -/- and HCT-116 p21 -/- cells. Cells were harvested and processed for cell cycle analysis by flow cytometry after treatment for 48 hr. The results are representative of three independent experiments.
    Figure Legend Snippet: Effects of DPA on cell cycle progression of HCT-116, HCT-116 p53 -/- and HCT-116 p21 -/- cells. Cells were harvested and processed for cell cycle analysis by flow cytometry after treatment for 48 hr. The results are representative of three independent experiments.

    Techniques Used: Cell Cycle Assay, Flow Cytometry, Cytometry

    36) Product Images from "Pathogenesis of the Erythroid Failure in Diamond Blackfan Anemia"

    Article Title: Pathogenesis of the Erythroid Failure in Diamond Blackfan Anemia

    Journal: British journal of haematology

    doi: 10.1111/j.1365-2141.2009.07993.x

    A , Ter119− fetal liver cells cultured without (control) or following infection with siLuciferase (GFP) or siRps19 for 6 days; Western analysis of protein expression using antibodies to p53, c-Myb or GAPDH. B , normalized ratio of p53:GAPDH, showing a modest 20% increase in p53 in siRPS19.2 compared with the siLuciferase empty vector control (n=3). C , normalized ratio of MYB:GAPDH, showing a marked decrease in MYB in siRPS19.2 compared with the siLuciferase control (n=3).
    Figure Legend Snippet: A , Ter119− fetal liver cells cultured without (control) or following infection with siLuciferase (GFP) or siRps19 for 6 days; Western analysis of protein expression using antibodies to p53, c-Myb or GAPDH. B , normalized ratio of p53:GAPDH, showing a modest 20% increase in p53 in siRPS19.2 compared with the siLuciferase empty vector control (n=3). C , normalized ratio of MYB:GAPDH, showing a marked decrease in MYB in siRPS19.2 compared with the siLuciferase control (n=3).

    Techniques Used: Cell Culture, Infection, Western Blot, Expressing, Plasmid Preparation

    37) Product Images from "STAR RNA-binding protein Quaking suppresses cancer via stabilization of specific miRNA"

    Article Title: STAR RNA-binding protein Quaking suppresses cancer via stabilization of specific miRNA

    Journal: Genes & Development

    doi: 10.1101/gad.189001.112

    QKI is a GBM tumor suppressor gene. ( A–C ) QKI was knocked down by shRNAs, and 5000 human GBM cells or 20,000 mouse astrocytes were plated per well in six-well plates for soft agar colony assay. Experiments were performed in triplicates. U87 (p53-WT) ( A ) and Hs683 (p53-null) ( B ) human GBM cell lines, as well as Ink4a/Arf −/− Pten −/− primary mouse astrocytes ( C ) transduced with two independent shRNAs (shQKI-1 and shQKI-2) against QKI showed increased soft agar colony growth relative to shGFP and noneffective shQKI control (shQKI-3). A Western blot showing QKI protein levels upon shRNA knockdown is presented in A for the human system and in C for the mouse system. The soft agar colony result is presented in B ; magnification 40×. (**) P
    Figure Legend Snippet: QKI is a GBM tumor suppressor gene. ( A–C ) QKI was knocked down by shRNAs, and 5000 human GBM cells or 20,000 mouse astrocytes were plated per well in six-well plates for soft agar colony assay. Experiments were performed in triplicates. U87 (p53-WT) ( A ) and Hs683 (p53-null) ( B ) human GBM cell lines, as well as Ink4a/Arf −/− Pten −/− primary mouse astrocytes ( C ) transduced with two independent shRNAs (shQKI-1 and shQKI-2) against QKI showed increased soft agar colony growth relative to shGFP and noneffective shQKI control (shQKI-3). A Western blot showing QKI protein levels upon shRNA knockdown is presented in A for the human system and in C for the mouse system. The soft agar colony result is presented in B ; magnification 40×. (**) P

    Techniques Used: Colony Assay, Transduction, Western Blot, shRNA

    QKI is a p53 target gene. ( A–C ) Qki was originally identified in mTerc −/− Atm −/− p53 −/− MEFs transduced with the inducible p53ER expression vector. To verify, Qki RNA ( A ) and protein ( C ) levels were measured and confirmed to be activated upon tamoxifen (Tam) treatment relative to ethanol (Etoh); p21 ( B ) served as positive control. RNA level was measured by qPCR. (*) P
    Figure Legend Snippet: QKI is a p53 target gene. ( A–C ) Qki was originally identified in mTerc −/− Atm −/− p53 −/− MEFs transduced with the inducible p53ER expression vector. To verify, Qki RNA ( A ) and protein ( C ) levels were measured and confirmed to be activated upon tamoxifen (Tam) treatment relative to ethanol (Etoh); p21 ( B ) served as positive control. RNA level was measured by qPCR. (*) P

    Techniques Used: Transduction, Expressing, Plasmid Preparation, Positive Control, Real-time Polymerase Chain Reaction

    QKI mechanism correlates with expression and genomic alterations in TCGA human GBM patient samples. ( A ) A model for the mechanism of QKI in GBM tumor suppression. While p53 can transcriptionally activate both QKI and miR-20a, QKI has an independent role of up-regulating miR-20a at least partially through stabilization. miR-20a in turn inhibits TGFβR2, an oncogene in gliomagenesis. p53, QKI, and miR-20a can additionally act on downstream targets other than those proposed in this model. ( B ) TP53 and QKI demonstrate epistatic relationships in TCGA genomic analysis. ( C ) An analysis for transcript expression correlation among 389 human GBM patients in TCGA. QKI is positively correlated with miR-20a. miR-20a is negatively correlated with TGFβR2.
    Figure Legend Snippet: QKI mechanism correlates with expression and genomic alterations in TCGA human GBM patient samples. ( A ) A model for the mechanism of QKI in GBM tumor suppression. While p53 can transcriptionally activate both QKI and miR-20a, QKI has an independent role of up-regulating miR-20a at least partially through stabilization. miR-20a in turn inhibits TGFβR2, an oncogene in gliomagenesis. p53, QKI, and miR-20a can additionally act on downstream targets other than those proposed in this model. ( B ) TP53 and QKI demonstrate epistatic relationships in TCGA genomic analysis. ( C ) An analysis for transcript expression correlation among 389 human GBM patients in TCGA. QKI is positively correlated with miR-20a. miR-20a is negatively correlated with TGFβR2.

    Techniques Used: Expressing, Activated Clotting Time Assay

    38) Product Images from "XIAP-associating factor 1, a transcriptional target of BRD7, contributes to endothelial cell senescence"

    Article Title: XIAP-associating factor 1, a transcriptional target of BRD7, contributes to endothelial cell senescence

    Journal: Oncotarget

    doi: 10.18632/oncotarget.6962

    Transcriptional regulation of XAF1 by BRD7 A. XAF1 was upregulated with BRD7 lentivirus or downregulated with BRD7 siRNAs in HMVECs. BRD7, CMPK2, ASPM, STIL, SQLE, PLAC8, RIOK2, XAF1, CFB, CBF4 and DTX3L expression levels were analyzed by real time-PCR. Each of the expression levels were normalized to GAPDH expression levels. B. Cells were transduced with BRD7 lentivirus or treated with 0, 20 or 100 ng/ml interferon-gamma (IFN-γ) for 24 h. XAF1, BRD7, IRF1, phospho-p53 and Ac-H3K9 protein levels were detected by Western blot analysis. C. Cells were transfected with BRD7 siRNAs and treated with IFN-γ for 24 h. XAF1, BRD7, IRF1 and phsopho-p53 protein levels were detected by Western blot analysis. D. Cells were transduced with BRD7 lentivirus or transfected with BRD7 siRNAs. Each of cells was treated with 4 Gy IR for 24 h. Chromatin immunoprecipitation (ChIP) assay was performed using the anti-BRD7 antibody and the immunoprecipitated DNA was amplified using primers for p53, IRF1 or XAF1. E. The cells were transfected with the luciferase reporter constructs containing the XAF1 promoter. XAF1 promoter activity in BRD7-expressed cells was determined by luciferase analysis. * p
    Figure Legend Snippet: Transcriptional regulation of XAF1 by BRD7 A. XAF1 was upregulated with BRD7 lentivirus or downregulated with BRD7 siRNAs in HMVECs. BRD7, CMPK2, ASPM, STIL, SQLE, PLAC8, RIOK2, XAF1, CFB, CBF4 and DTX3L expression levels were analyzed by real time-PCR. Each of the expression levels were normalized to GAPDH expression levels. B. Cells were transduced with BRD7 lentivirus or treated with 0, 20 or 100 ng/ml interferon-gamma (IFN-γ) for 24 h. XAF1, BRD7, IRF1, phospho-p53 and Ac-H3K9 protein levels were detected by Western blot analysis. C. Cells were transfected with BRD7 siRNAs and treated with IFN-γ for 24 h. XAF1, BRD7, IRF1 and phsopho-p53 protein levels were detected by Western blot analysis. D. Cells were transduced with BRD7 lentivirus or transfected with BRD7 siRNAs. Each of cells was treated with 4 Gy IR for 24 h. Chromatin immunoprecipitation (ChIP) assay was performed using the anti-BRD7 antibody and the immunoprecipitated DNA was amplified using primers for p53, IRF1 or XAF1. E. The cells were transfected with the luciferase reporter constructs containing the XAF1 promoter. XAF1 promoter activity in BRD7-expressed cells was determined by luciferase analysis. * p

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Transduction, Western Blot, Transfection, Chromatin Immunoprecipitation, Immunoprecipitation, Amplification, Luciferase, Construct, Activity Assay

    Effects of XAF1 on the upregulation of cellular senescence in young HMVECs A. Young cells were transduced with XAF1 or negative control lentiviruses and incubated for 3 days. XAF1 mRNA expression levels were measured by semi-quantitative PCR and XAF1, cyclin A, p53 and p21 protein levels were detected by Western blot analysis. B. Cell proliferation was measured by cell counting and C. the percentages of SA-β-gal positive cells were analyzed. * p
    Figure Legend Snippet: Effects of XAF1 on the upregulation of cellular senescence in young HMVECs A. Young cells were transduced with XAF1 or negative control lentiviruses and incubated for 3 days. XAF1 mRNA expression levels were measured by semi-quantitative PCR and XAF1, cyclin A, p53 and p21 protein levels were detected by Western blot analysis. B. Cell proliferation was measured by cell counting and C. the percentages of SA-β-gal positive cells were analyzed. * p

    Techniques Used: Transduction, Negative Control, Incubation, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Cell Counting

    Effects of p53 or p16 knockdown on cell growth arrest induced by XAF1 A. Cells were transduced with p53- or p16-shRNA retroviruses and incubated for 2 days. P53 and p16 knockdown was confirmed by RT-PCR analysis. The p53- or p16-downregulated cells were transduced with XAF1 or negative control lentiviruses and incubated for 2 days. Cell proliferation was measured by B. cell counting for 2 days and C. SA-β-gal staining (100x). Values are expressed as the mean ± SD of three independent experiments. Representative data from three independent experiments are shown. * p
    Figure Legend Snippet: Effects of p53 or p16 knockdown on cell growth arrest induced by XAF1 A. Cells were transduced with p53- or p16-shRNA retroviruses and incubated for 2 days. P53 and p16 knockdown was confirmed by RT-PCR analysis. The p53- or p16-downregulated cells were transduced with XAF1 or negative control lentiviruses and incubated for 2 days. Cell proliferation was measured by B. cell counting for 2 days and C. SA-β-gal staining (100x). Values are expressed as the mean ± SD of three independent experiments. Representative data from three independent experiments are shown. * p

    Techniques Used: Transduction, shRNA, Incubation, Reverse Transcription Polymerase Chain Reaction, Negative Control, Cell Counting, Staining

    Increase in XAF1 expression in cellular senescence A. XAF1 protein expression levels XAF1 were analyzed by Western blotting with antibodies against XAF1 and GAPDH (loading control) in aged HMVECs. The levels of cyclin A, phospho-p53(S15), p53, p21 and β-actin proteins were detected by Western blot analysis. The figure shows representative data from three independent experiments. B. Percentages of SA-β-gal positive cells analyzed in young and old HMVECs. ** p
    Figure Legend Snippet: Increase in XAF1 expression in cellular senescence A. XAF1 protein expression levels XAF1 were analyzed by Western blotting with antibodies against XAF1 and GAPDH (loading control) in aged HMVECs. The levels of cyclin A, phospho-p53(S15), p53, p21 and β-actin proteins were detected by Western blot analysis. The figure shows representative data from three independent experiments. B. Percentages of SA-β-gal positive cells analyzed in young and old HMVECs. ** p

    Techniques Used: Expressing, Western Blot

    39) Product Images from "TGF-β1/IL-11/MEK/ERK signaling mediates senescence-associated pulmonary fibrosis in a stress-induced premature senescence model of Bmi-1 deficiency"

    Article Title: TGF-β1/IL-11/MEK/ERK signaling mediates senescence-associated pulmonary fibrosis in a stress-induced premature senescence model of Bmi-1 deficiency

    Journal: Experimental & Molecular Medicine

    doi: 10.1038/s12276-019-0371-7

    NAC treatment inhibited cell senescence and TIME signaling in Bmi-1 −/− more than in Bmi-1 −/− p16 −/− pulmonary fibroblasts. Pulmonary fibroblasts from 7-week-old WT, Bmi-1 −/− , Bmi-1 −/− p16 −/− , and NAC-treated Bmi-1 −/− ( Bmi-1 −/− + NAC) mice. a Representative micrographs of cells stained cytochemically for SA-β-gal. b Percentage of SA-β-gal-positive areas relative to the total areas. c Third-passage fibroblast proliferation was determined by CCK-8 assays and spectrophotometry at 450 nm. Cell number was determined at the indicated hours relative to the cell number at hour 0. d TGF-β1 and IL-11 concentrations were detected in conditioned medium (CM) by ELISA and assessed by densitometric analysis relative to that of WT CM. e , f Western blots for mature TGF-β1, IL-11, p19, p53, p21, α-SMA, p16, pERK1/2 (Thr202/Tyr204), and ERK1/2. β-actin was used as the loading control. g Protein expression relative to β-actin was assessed by densitometric analysis. h Representative micrographs showing immunofluorescence for IL-11 and p53, with DAPI staining the nuclei. i Percentage of IL-11-positive areas (in p53-positive cells) or p53-positive areas relative to the total area assessed. Six biological replicates were used per experiment. Values are the means ± SEM of six determinations. * P
    Figure Legend Snippet: NAC treatment inhibited cell senescence and TIME signaling in Bmi-1 −/− more than in Bmi-1 −/− p16 −/− pulmonary fibroblasts. Pulmonary fibroblasts from 7-week-old WT, Bmi-1 −/− , Bmi-1 −/− p16 −/− , and NAC-treated Bmi-1 −/− ( Bmi-1 −/− + NAC) mice. a Representative micrographs of cells stained cytochemically for SA-β-gal. b Percentage of SA-β-gal-positive areas relative to the total areas. c Third-passage fibroblast proliferation was determined by CCK-8 assays and spectrophotometry at 450 nm. Cell number was determined at the indicated hours relative to the cell number at hour 0. d TGF-β1 and IL-11 concentrations were detected in conditioned medium (CM) by ELISA and assessed by densitometric analysis relative to that of WT CM. e , f Western blots for mature TGF-β1, IL-11, p19, p53, p21, α-SMA, p16, pERK1/2 (Thr202/Tyr204), and ERK1/2. β-actin was used as the loading control. g Protein expression relative to β-actin was assessed by densitometric analysis. h Representative micrographs showing immunofluorescence for IL-11 and p53, with DAPI staining the nuclei. i Percentage of IL-11-positive areas (in p53-positive cells) or p53-positive areas relative to the total area assessed. Six biological replicates were used per experiment. Values are the means ± SEM of six determinations. * P

    Techniques Used: Mouse Assay, Staining, CCK-8 Assay, Spectrophotometry, Enzyme-linked Immunosorbent Assay, Western Blot, Expressing, Immunofluorescence

    NAC treatment inhibited cell senescence, and TIME signaling mediated the EMT in Bmi-1 −/− cells more than in Bmi-1 −/− p16 −/− AT2 cells. Pulmonary AT2 cells from 7-week-old WT, Bmi-1 −/− , Bmi-1 −/− p16 −/− , and NAC-treated Bmi-1 −/− ( Bmi-1 −/− + NAC) mice. a Representative micrographs of cells stained cytochemically for SA-β-gal. b Percentage of SA-β-gal-positive areas relative to the total. c Second-passage AT2 cell proliferation were determined by CCK-8 assays, and spectrophotometry at 450 nm. Cell number was determined at the indicated hours relative to the cell number at hour 0. d Western blots for SFTPC, α-SMA, pERK1/2 (Thr202/Tyr204), ERK1/2, p16, p53, p21, mature TGF-β1, IL-11, and Snail. β-actin was used as the loading control. e Protein expression relative to β-actin was assessed by densitometric analysis. f Pulmonary AT2 cells were treated with TGF-β1 (5 ng/ml, 48 h), TGF-β1 (5 ng/ml, 48 h), and anti-IL-11 antibody (2 μg/ml, 48 h), IL-11 (5 ng/ml, 48 h), or IL-11 (5 ng/ml, 48 h) and the MEK inhibitor (PD98059) (10 μ m , 48 h). Western blots for SFTPC, α-SMA, pERK1/2 (Thr202/Tyr204), ERK1/2, p16, p53, and p21. β-actin was used as the loading control. g Protein expression relative to β-actin was assessed by densitometric analysis. h Representative micrographs of cells immunofluorescently stained for α-SMA and SFTPC, with DAPI staining the nuclei. i The percentage of α-SMA- or SFTPC-positive areas was relative to the total. j WT AT2 cells treated with CM from third-passage pulmonary fibroblasts from the WT, Bmi-1 −/− , Bmi-1 −/− p16 −/− or NAC-treated Bmi-1 −/− ( Bmi-1 −/− + NAC) groups. k The percentage of α-SMA- or SFTPC-positive areas relative to the total. Six biological replicates were used per experiment. Values are the means ± SEM of six determinations. * P
    Figure Legend Snippet: NAC treatment inhibited cell senescence, and TIME signaling mediated the EMT in Bmi-1 −/− cells more than in Bmi-1 −/− p16 −/− AT2 cells. Pulmonary AT2 cells from 7-week-old WT, Bmi-1 −/− , Bmi-1 −/− p16 −/− , and NAC-treated Bmi-1 −/− ( Bmi-1 −/− + NAC) mice. a Representative micrographs of cells stained cytochemically for SA-β-gal. b Percentage of SA-β-gal-positive areas relative to the total. c Second-passage AT2 cell proliferation were determined by CCK-8 assays, and spectrophotometry at 450 nm. Cell number was determined at the indicated hours relative to the cell number at hour 0. d Western blots for SFTPC, α-SMA, pERK1/2 (Thr202/Tyr204), ERK1/2, p16, p53, p21, mature TGF-β1, IL-11, and Snail. β-actin was used as the loading control. e Protein expression relative to β-actin was assessed by densitometric analysis. f Pulmonary AT2 cells were treated with TGF-β1 (5 ng/ml, 48 h), TGF-β1 (5 ng/ml, 48 h), and anti-IL-11 antibody (2 μg/ml, 48 h), IL-11 (5 ng/ml, 48 h), or IL-11 (5 ng/ml, 48 h) and the MEK inhibitor (PD98059) (10 μ m , 48 h). Western blots for SFTPC, α-SMA, pERK1/2 (Thr202/Tyr204), ERK1/2, p16, p53, and p21. β-actin was used as the loading control. g Protein expression relative to β-actin was assessed by densitometric analysis. h Representative micrographs of cells immunofluorescently stained for α-SMA and SFTPC, with DAPI staining the nuclei. i The percentage of α-SMA- or SFTPC-positive areas was relative to the total. j WT AT2 cells treated with CM from third-passage pulmonary fibroblasts from the WT, Bmi-1 −/− , Bmi-1 −/− p16 −/− or NAC-treated Bmi-1 −/− ( Bmi-1 −/− + NAC) groups. k The percentage of α-SMA- or SFTPC-positive areas relative to the total. Six biological replicates were used per experiment. Values are the means ± SEM of six determinations. * P

    Techniques Used: Mouse Assay, Staining, CCK-8 Assay, Spectrophotometry, Western Blot, Expressing

    NAC treatment improved pulmonary cell senescence, DNA damage, and SASP in Bmi-1 −/− mice more than in Bmi-1 −/− p16 −/− mice. a Representative micrographs of paraffin-embedded pulmonary sections stained for senescence-associated β-galactosidase (SA-β-gal), stained immunohistochemically for p53, 8-hydroxyguanosine (8-OHdG), IL-1β, IL-6, and TNF-α, and stained immunofluorescently for CD3 and F4/80. SA-β-gal-, p53-, 8-OHdG-, CD3-, or F4/80-positive cells are indicated by arrows. b Percentage of cells positive for SA-β-gal, p53, 8-OHdG, CD3, F4/80, IL-1β, IL-6, and TNF-α or with positive area relative to the total cells or area. c Bmi-1 , p16 , IL-1a , IL-1β , IL-6 , and TNF-α mRNA levels in lungs by real-time RT-PCR, calculated as the ratio to β-actin mRNA and expressed relative to that of WT. d Western blots of pulmonary extracts showing p16, p19, p53, p21, and 8-OHdG. β-actin was used as the loading control. e Protein levels relative to β-actin were assessed by densitometric analysis. Six mice per group were used for experiments. Values are the mean ± SEM from six determinations per group. * P
    Figure Legend Snippet: NAC treatment improved pulmonary cell senescence, DNA damage, and SASP in Bmi-1 −/− mice more than in Bmi-1 −/− p16 −/− mice. a Representative micrographs of paraffin-embedded pulmonary sections stained for senescence-associated β-galactosidase (SA-β-gal), stained immunohistochemically for p53, 8-hydroxyguanosine (8-OHdG), IL-1β, IL-6, and TNF-α, and stained immunofluorescently for CD3 and F4/80. SA-β-gal-, p53-, 8-OHdG-, CD3-, or F4/80-positive cells are indicated by arrows. b Percentage of cells positive for SA-β-gal, p53, 8-OHdG, CD3, F4/80, IL-1β, IL-6, and TNF-α or with positive area relative to the total cells or area. c Bmi-1 , p16 , IL-1a , IL-1β , IL-6 , and TNF-α mRNA levels in lungs by real-time RT-PCR, calculated as the ratio to β-actin mRNA and expressed relative to that of WT. d Western blots of pulmonary extracts showing p16, p19, p53, p21, and 8-OHdG. β-actin was used as the loading control. e Protein levels relative to β-actin were assessed by densitometric analysis. Six mice per group were used for experiments. Values are the mean ± SEM from six determinations per group. * P

    Techniques Used: Mouse Assay, Staining, Quantitative RT-PCR, Western Blot

    40) Product Images from "Silencing of zinc finger protein 703 inhibits medullary thyroid carcinoma cell proliferation in vitro and in vivo"

    Article Title: Silencing of zinc finger protein 703 inhibits medullary thyroid carcinoma cell proliferation in vitro and in vivo

    Journal: Oncology Letters

    doi: 10.3892/ol.2019.11153

    ZNF703 silencing regulates the levels of pAKT 473 and p53 protein in vitro and in vivo . (A) Akt, pAKT 473 and p53 protein expression in TT cells, with or without ZNF703 silencing, were determined by western blotting. Each data point represents the mean ± SD of three independent experiments. (B) Akt, pAKT 473 and p53 protein expression in xenograft tumors, with or without ZNF703 silencing, were determined by western blotting. Each data point represents the mean ± SD of six xenograft tumors. *P
    Figure Legend Snippet: ZNF703 silencing regulates the levels of pAKT 473 and p53 protein in vitro and in vivo . (A) Akt, pAKT 473 and p53 protein expression in TT cells, with or without ZNF703 silencing, were determined by western blotting. Each data point represents the mean ± SD of three independent experiments. (B) Akt, pAKT 473 and p53 protein expression in xenograft tumors, with or without ZNF703 silencing, were determined by western blotting. Each data point represents the mean ± SD of six xenograft tumors. *P

    Techniques Used: In Vitro, In Vivo, Expressing, Western Blot

    Related Articles

    Functional Assay:

    Article Title: SOX11 and TP53 add prognostic information to MIPI in a homogenously treated cohort of mantle cell lymphoma – a Nordic Lymphoma Group study
    Article Snippet: A positive correlation between SOX11 and CCND1 (P = 0·006) was seen, in contrast to previous data, where SOX11 expression was found to be independent of the t(11;14) translocation ( ). .. Blastoid morphology (P < 0·002), TP53 (P < 0·001) and MKI67 (P = 0·006) showed a negative correlation to SOX11, indicating that SOX11high may identify patients with lower proliferation, non-blastoid morphology and functional TP53. .. Prognostic significance of SOX11, TP53, MKI67, CCND1, blastoid morphology and MIPI To determine the prognostic significance of relevant molecular and clinicopathological parameters, Cox univariate analyses were performed.

    Immunoprecipitation:

    Article Title: Tumor-associated mutant p53 promotes cancer cell survival upon glutamine deprivation through p21 induction
    Article Snippet: After centrifugation, cell supernatant was diluted in immunoprecipitation buffer with protease inhibitors and pre-cleared with Salmon Sperm DNA/ Protein A Agarose for 1 hour at 4°C with rotation. .. 1 µg of p53 (DO-1, Santa Cruz) or IgG antibody was used for each overnight immunoprecipitation with rotation at 4°C. .. Binding sites of p53 were amplified by 30–35 cycles of PCR using Hotstart Taq DNA polymerase (Bioneer).

    Plasmid Preparation:

    Article Title: Antitumor Activity and Induction of TP53-Dependent Apoptosis toward Ovarian Clear Cell Adenocarcinoma by the Dual PI3K/mTOR Inhibitor DS-7423
    Article Snippet: The effect of DS-7423 on the transcriptional activity of TP53 was also examined by luciferase assays in ES-2 cells with mutations in TP53 . .. The cells were treated with DS-7423 for 24 h at the indicated doses, and then cotransfected with both pp53-TA-luc plasmid (containing TP53 binding sites) and a plasmid that encodes TP53. .. The relative luciferase activity of TP53 was significantly enhanced by DS-7423 in a dose-dependent manner ( ).

    Binding Assay:

    Article Title: Antitumor Activity and Induction of TP53-Dependent Apoptosis toward Ovarian Clear Cell Adenocarcinoma by the Dual PI3K/mTOR Inhibitor DS-7423
    Article Snippet: The effect of DS-7423 on the transcriptional activity of TP53 was also examined by luciferase assays in ES-2 cells with mutations in TP53 . .. The cells were treated with DS-7423 for 24 h at the indicated doses, and then cotransfected with both pp53-TA-luc plasmid (containing TP53 binding sites) and a plasmid that encodes TP53. .. The relative luciferase activity of TP53 was significantly enhanced by DS-7423 in a dose-dependent manner ( ).

    Formalin-fixed Paraffin-Embedded:

    Article Title: Epithelial PIK3R1 (p85) and TP53 Regulate Survivin Expression during Adaptation to Ileocecal Resection
    Article Snippet: .. Formalin-fixed, paraffin-embedded sections were stained for BrdU (MPL International, Woburn, MA), Ki-67 (Dako, Glostrup, Denmark), survivin (Novus Biologicals, Littleton, CO), and TP53 (Santa Cruz Biotechnology, Dallas, TX) using standard IHC methods. .. Tissues were counterstained with hematoxylin (except for TP53 staining).

    Staining:

    Article Title: Epithelial PIK3R1 (p85) and TP53 Regulate Survivin Expression during Adaptation to Ileocecal Resection
    Article Snippet: .. Formalin-fixed, paraffin-embedded sections were stained for BrdU (MPL International, Woburn, MA), Ki-67 (Dako, Glostrup, Denmark), survivin (Novus Biologicals, Littleton, CO), and TP53 (Santa Cruz Biotechnology, Dallas, TX) using standard IHC methods. .. Tissues were counterstained with hematoxylin (except for TP53 staining).

    Immunohistochemistry:

    Article Title: Epithelial PIK3R1 (p85) and TP53 Regulate Survivin Expression during Adaptation to Ileocecal Resection
    Article Snippet: .. Formalin-fixed, paraffin-embedded sections were stained for BrdU (MPL International, Woburn, MA), Ki-67 (Dako, Glostrup, Denmark), survivin (Novus Biologicals, Littleton, CO), and TP53 (Santa Cruz Biotechnology, Dallas, TX) using standard IHC methods. .. Tissues were counterstained with hematoxylin (except for TP53 staining).

    Incubation:

    Article Title: Silencing microRNA-143 protects the integrity of the blood-brain barrier: implications for methamphetamine abuse
    Article Snippet: Western blot analysis Proteins were extracted in RIPA lysis buffer (Beyotime, Shanghai, China), separated on sodium dodecyl sulfate polyacrylamide gels (8% and 12%) and electrophoretically transferred onto polyvinylidene fluoride membranes. .. The membranes were blocked with 5% non-fat dry milk in Tris-buffered saline with Tween-20, probed with antibodies recognizing p-ERK/ERK (1:1,000, Cell Signaling, #9101S/#9107S), p-JNK(1:1,000, Santa Cruz, sc-6254)/JNK(1:1,000, Cell Signaling, #9252S), p-p38/p38(1:1,000, Cell Signaling, #9211S/#9212S), p-AKT/AKT (1:1,000, Cell Signaling, #9271S/#9272S), NF-κB p65 (1:1,000, Cell Signaling, #3033S), STAT3 (1:1,000, Cell Signaling, #12640S), histone H3 (1:1,000, Cell Signaling, #9715S), claudin-5 (1:1,000, Abcam, ab15106), occludin (1:1,000, Life Technology, #33–1500), ZO-1 (1:1,000, Life Technology, #402300), p53 (1:1,000, Santa Cruz, sc-6243), PUMA (1:1,000, Santa Cruz, sc-28226) and β-actin (1:1,000, Bioworld, BS6007M) overnight at 4 °C, and then incubated with a horseradish peroxidase-conjugated goat anti-mouse/rabbit IgG secondary antibody (1:2,000, Cell Signaling, #7076P2/#7074P2). .. A Microchemi 4.2® (DNR, Israel) digital image scanner was used for detection, and the band intensity was quantified using ImageJ software (NIH).

    Expressing:

    Article Title: A regulatory circuitry comprising TP53, miR-29 family, and SETDB1 in non-small cell lung cancer
    Article Snippet: Next, the scatter plot and the Pearson correlation analysis demonstrated that the expression levels of miR-29a , -29b , and -29c were negatively correlated with SETDB1 mRNA levels but positively correlated with TP53 mRNA levels, respectively ( D–F). .. These data suggested that the TP53, miR-29s , and SETDB1 appear to modulate the expression of each other and form a self-regulated circle loop in NSCLC. .. MiR-29s affect the H3K9 methylation status in NSCLC Our newly identified miR-29s /SETDB1/TP53 regulatory circuitry indicated that miR-29s positively regulate TP53 expression via directly targetting SETDB1.

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    Santa Cruz Biotechnology p53 protein
    Expression of apoptosis modulators BCL-2, BAX and BBC3 during chemotherapy . The ratio of BCL2/BAX or BCL2/BBC3 mRNA was determined after Agilent Human Whole Genome Oligo Microarray analysis ( A ). Ratios were observed to decrease in the first hours post induction treatment, indicating an increase in pro-apoptotic gene expression. Ratios varied greatly between patients and low baseline ratios were associated with better response to treatment (Additional File 1 ). Ratios were normalized and mean presented with standard deviation (error bars) ( B ), indicating early pro-apoptotic alteration in BCL2/BAX mRNA. Two patients (P3 and P8) were treated with anthracycline ex vivo and protein expression analyzed using Western blots ( C ). The ex vivo response to anthracycline included increased stabilization of <t>p53</t> followed by regulation of p53 target gene expression in concordance with in vivo observations.
    P53 Protein, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    p53 protein - by Bioz Stars, 2021-06
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    99
    Santa Cruz Biotechnology anti p53
    SYK inhibition by entospletinib (E) and fostamatinib (F) reduces <t>p53</t> level (D). HCT116 and HT1080 cells were treated with 1.5 μM doxorubicin or 1 μM doxorubicin (D) and 1 μM entospletinib or 0.5 μM fostamatinib for 24 hours. Entospletinib and fostamatinib reduced p53 levels in both cell lines. Error bars showed p53 and SYK quantification from different experiments.
    Anti P53, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti p53/product/Santa Cruz Biotechnology
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti p53 - by Bioz Stars, 2021-06
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    N/A
    Gene Silencers generally consist of pools of three to five target specific 19 25 nucleotide sequences in length For independent verification of p53 gene silencing results individual duplex components or
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    Image Search Results


    Effect of p53 knock-down on Atox1 expression in MEF Atox1+/+ cells. (a) Confocal images of MEF Atox1+/+ cells after KD of p53 using p53-siRNA. Cells were seeded on glass coverslips, treated with 20 pmol p53-siRNA for 48 h and fixed with 4% PFA. p53 (green)

    Journal: Journal of biological inorganic chemistry : JBIC : a publication of the Society of Biological Inorganic Chemistry

    Article Title: Roles of Atox1 and p53 in the trafficking of copper-64 to tumor cell nuclei: implications for cancer therapy

    doi: 10.1007/s00775-013-1087-0

    Figure Lengend Snippet: Effect of p53 knock-down on Atox1 expression in MEF Atox1+/+ cells. (a) Confocal images of MEF Atox1+/+ cells after KD of p53 using p53-siRNA. Cells were seeded on glass coverslips, treated with 20 pmol p53-siRNA for 48 h and fixed with 4% PFA. p53 (green)

    Article Snippet: MEF cells were seeded in 6-well plates or on glass cover slips in 24-well plates to attain 20-30% confluence after 24 h. The transfection was done with p53 siRNA (Santa Cruz Biotechnology, Dallas, TX) using RNAiMAX lipofectamine reagent (Invitrogen, Grand Island, NY) according to the manufacturer's instructions (20 pmol siRNA).

    Techniques: Expressing

    Expression of apoptosis modulators BCL-2, BAX and BBC3 during chemotherapy . The ratio of BCL2/BAX or BCL2/BBC3 mRNA was determined after Agilent Human Whole Genome Oligo Microarray analysis ( A ). Ratios were observed to decrease in the first hours post induction treatment, indicating an increase in pro-apoptotic gene expression. Ratios varied greatly between patients and low baseline ratios were associated with better response to treatment (Additional File 1 ). Ratios were normalized and mean presented with standard deviation (error bars) ( B ), indicating early pro-apoptotic alteration in BCL2/BAX mRNA. Two patients (P3 and P8) were treated with anthracycline ex vivo and protein expression analyzed using Western blots ( C ). The ex vivo response to anthracycline included increased stabilization of p53 followed by regulation of p53 target gene expression in concordance with in vivo observations.

    Journal: BMC Cancer

    Article Title: Genes of cell-cell interactions, chemotherapy detoxification and apoptosis are induced during chemotherapy of acute myeloid leukemia

    doi: 10.1186/1471-2407-9-77

    Figure Lengend Snippet: Expression of apoptosis modulators BCL-2, BAX and BBC3 during chemotherapy . The ratio of BCL2/BAX or BCL2/BBC3 mRNA was determined after Agilent Human Whole Genome Oligo Microarray analysis ( A ). Ratios were observed to decrease in the first hours post induction treatment, indicating an increase in pro-apoptotic gene expression. Ratios varied greatly between patients and low baseline ratios were associated with better response to treatment (Additional File 1 ). Ratios were normalized and mean presented with standard deviation (error bars) ( B ), indicating early pro-apoptotic alteration in BCL2/BAX mRNA. Two patients (P3 and P8) were treated with anthracycline ex vivo and protein expression analyzed using Western blots ( C ). The ex vivo response to anthracycline included increased stabilization of p53 followed by regulation of p53 target gene expression in concordance with in vivo observations.

    Article Snippet: The p53 protein was detected using Bp53-12 monoclonal antibody, BAX protein was detected using primary 2D2 antibody, BCL-2 protein was detected using primary ΔC21 antibody and procaspase-3 protein was detected using primary E-8 antibody (Santa Cruz Biotechnology, Santa Cruz, CA) and actin protein was detected using primary AC-15 antibody (Abcam, Cambridge, UK).

    Techniques: Expressing, Microarray, Standard Deviation, Ex Vivo, Western Blot, In Vivo

    DNA microarray analysis in AML cells during in vivo chemotherapy . AML blast viability was investigated in four patients sampled during in vivo chemotherapy. The expression of apoptosis related proteins were detected by Western blotting (P3, P4) ( A ). Expression of the pro-apoptotic protein BAX increased during therapy, suggesting initiation of cell death. However, no cleavage of the executioner protease pro-caspase-3 could be detected and this is in agreement with the flowcytometric analysis in (B). Apoptosis was measured using flowcytometric detection of Annexin-V FITC and propidium iodide staining (P1, P3) as described in Methods. No signs of apoptosis could be detected in the cells during the first six hours of therapy in either patient ( B ). Similar viability (~80%) was present at 18 h determined by analysis of nuclear morphology and cell scattering (data not shown). Early changes in gene expression of p53-associated genes were detected already 2–4 hours following induction therapy ( C ). The profiles of 27 of the 31 p53 associated genes are shown. Late response gene expression of p53-inducible genes was detected 18–24 hours after treatment induction ( D ). For abbreviations see Additional File 2 . The dendrogram and heat maps show a Eucledian two-way cluster analysis based on the most consistently upregulated genes. Thus, genes that cluster together have a similar expression profile as a response to induction therapy. In the diagram the relative mRNA levels in the blasts before therapy are colored in green, and upregulated genes following standard chemotherapy treatment are shown in violet according to the color scale below.

    Journal: BMC Cancer

    Article Title: Genes of cell-cell interactions, chemotherapy detoxification and apoptosis are induced during chemotherapy of acute myeloid leukemia

    doi: 10.1186/1471-2407-9-77

    Figure Lengend Snippet: DNA microarray analysis in AML cells during in vivo chemotherapy . AML blast viability was investigated in four patients sampled during in vivo chemotherapy. The expression of apoptosis related proteins were detected by Western blotting (P3, P4) ( A ). Expression of the pro-apoptotic protein BAX increased during therapy, suggesting initiation of cell death. However, no cleavage of the executioner protease pro-caspase-3 could be detected and this is in agreement with the flowcytometric analysis in (B). Apoptosis was measured using flowcytometric detection of Annexin-V FITC and propidium iodide staining (P1, P3) as described in Methods. No signs of apoptosis could be detected in the cells during the first six hours of therapy in either patient ( B ). Similar viability (~80%) was present at 18 h determined by analysis of nuclear morphology and cell scattering (data not shown). Early changes in gene expression of p53-associated genes were detected already 2–4 hours following induction therapy ( C ). The profiles of 27 of the 31 p53 associated genes are shown. Late response gene expression of p53-inducible genes was detected 18–24 hours after treatment induction ( D ). For abbreviations see Additional File 2 . The dendrogram and heat maps show a Eucledian two-way cluster analysis based on the most consistently upregulated genes. Thus, genes that cluster together have a similar expression profile as a response to induction therapy. In the diagram the relative mRNA levels in the blasts before therapy are colored in green, and upregulated genes following standard chemotherapy treatment are shown in violet according to the color scale below.

    Article Snippet: The p53 protein was detected using Bp53-12 monoclonal antibody, BAX protein was detected using primary 2D2 antibody, BCL-2 protein was detected using primary ΔC21 antibody and procaspase-3 protein was detected using primary E-8 antibody (Santa Cruz Biotechnology, Santa Cruz, CA) and actin protein was detected using primary AC-15 antibody (Abcam, Cambridge, UK).

    Techniques: Microarray, In Vivo, Expressing, Western Blot, Staining

    SYK inhibition by entospletinib (E) and fostamatinib (F) reduces p53 level (D). HCT116 and HT1080 cells were treated with 1.5 μM doxorubicin or 1 μM doxorubicin (D) and 1 μM entospletinib or 0.5 μM fostamatinib for 24 hours. Entospletinib and fostamatinib reduced p53 levels in both cell lines. Error bars showed p53 and SYK quantification from different experiments.

    Journal: Journal of Cell Death

    Article Title: Spleen Tyrosine Kinase Inhibition Modulates p53 Activity

    doi: 10.1177/1179066017731564

    Figure Lengend Snippet: SYK inhibition by entospletinib (E) and fostamatinib (F) reduces p53 level (D). HCT116 and HT1080 cells were treated with 1.5 μM doxorubicin or 1 μM doxorubicin (D) and 1 μM entospletinib or 0.5 μM fostamatinib for 24 hours. Entospletinib and fostamatinib reduced p53 levels in both cell lines. Error bars showed p53 and SYK quantification from different experiments.

    Article Snippet: Primary antibodies used were, anti-p53 (DO-1; Santa Cruz: sc-129), anti-SYK (N-19): sc-1077, anti-β-actin (ab8227; Abcam).

    Techniques: Inhibition

    hScrib regulates the expression of HPV-18 E6 in HeLa cells. Panel A. HPV-positive HeLa cells were transfected with siRNA Luciferase or siRNA against the indicated E6 PDZ substrates. Cells were grown for 72 h prior to harvesting and the expression patterns of HPV-18 E6, hDlg, hScrib, TIP2, p53, E6AP and α-actinin (to monitor the protein loading) were assessed by western blot analysis. Note that Lanes 1 and 2 have been spliced but all samples were run on the same gel. Panel B. Band intensities were determined using the OptiQuant quantification program. E6 levels were normalized to 100% relative to siLuciferase-transfected HeLa cells. Standard deviations are also shown. Panel C. The silencing of hScrib was performed as in A but using two different siRNAs specific for hScrib. The expression levels of HPV-18 E6, hScrib and α-actinin to monitor the protein loading, were assessed by western blot analysis. Panel D. Strips showing levels of HPV16 E6 detection in CaSki and SiHa cells following transfection with siRNA luciferase (si Luc) or si Scrib. Arrows indicate the position of the internal positive control and the HPV-16 E6 specific band. The bottom panels show the quantification of the band intensities.

    Journal: Papillomavirus Research

    Article Title: The high-risk HPV E6 target scribble (hScrib) is required for HPV E6 expression in cervical tumour-derived cell lines

    doi: 10.1016/j.pvr.2016.04.001

    Figure Lengend Snippet: hScrib regulates the expression of HPV-18 E6 in HeLa cells. Panel A. HPV-positive HeLa cells were transfected with siRNA Luciferase or siRNA against the indicated E6 PDZ substrates. Cells were grown for 72 h prior to harvesting and the expression patterns of HPV-18 E6, hDlg, hScrib, TIP2, p53, E6AP and α-actinin (to monitor the protein loading) were assessed by western blot analysis. Note that Lanes 1 and 2 have been spliced but all samples were run on the same gel. Panel B. Band intensities were determined using the OptiQuant quantification program. E6 levels were normalized to 100% relative to siLuciferase-transfected HeLa cells. Standard deviations are also shown. Panel C. The silencing of hScrib was performed as in A but using two different siRNAs specific for hScrib. The expression levels of HPV-18 E6, hScrib and α-actinin to monitor the protein loading, were assessed by western blot analysis. Panel D. Strips showing levels of HPV16 E6 detection in CaSki and SiHa cells following transfection with siRNA luciferase (si Luc) or si Scrib. Arrows indicate the position of the internal positive control and the HPV-16 E6 specific band. The bottom panels show the quantification of the band intensities.

    Article Snippet: The following antibodies were purchased from Santa Cruz Biotechnology: mouse monoclonal anti-p53 (DO-1), mouse monoclonal anti-α-actinin (H-2), mouse monoclonal anti-Dlg (2D11), goat polyclonal anti-Scribble (C-20), goat polyclonal anti-TIP2 (N-19), rabbit polyclonal anti-E-cadherin (H-108) and mouse monoclonal anti-vimentin (V-9).

    Techniques: Expressing, Transfection, Luciferase, Western Blot, Positive Control

    Loss of hScrib does not affect HPV-18 E6 protein stability. Panel A. HeLa cells were transfected with siRNA against Luciferase or siRNA against hScrib, 72 h after transfection, cells were treated with cycloheximide for 5 different time points: 0, 15, 30, 60 and 120 minutes prior to harvesting. The expression levels of HPV-18 E6, p53, hScrib, and α-actinin to monitor the protein loading, were assessed by western blot. The collated results from 3 independent experiments are shown in panel B. Band intensities were determined using the OptiQuant quantification program. The E6 levels in siLuciferase and siScrib transfected cells were normalized to 100% at time 0. Standard deviations are also shown.

    Journal: Papillomavirus Research

    Article Title: The high-risk HPV E6 target scribble (hScrib) is required for HPV E6 expression in cervical tumour-derived cell lines

    doi: 10.1016/j.pvr.2016.04.001

    Figure Lengend Snippet: Loss of hScrib does not affect HPV-18 E6 protein stability. Panel A. HeLa cells were transfected with siRNA against Luciferase or siRNA against hScrib, 72 h after transfection, cells were treated with cycloheximide for 5 different time points: 0, 15, 30, 60 and 120 minutes prior to harvesting. The expression levels of HPV-18 E6, p53, hScrib, and α-actinin to monitor the protein loading, were assessed by western blot. The collated results from 3 independent experiments are shown in panel B. Band intensities were determined using the OptiQuant quantification program. The E6 levels in siLuciferase and siScrib transfected cells were normalized to 100% at time 0. Standard deviations are also shown.

    Article Snippet: The following antibodies were purchased from Santa Cruz Biotechnology: mouse monoclonal anti-p53 (DO-1), mouse monoclonal anti-α-actinin (H-2), mouse monoclonal anti-Dlg (2D11), goat polyclonal anti-Scribble (C-20), goat polyclonal anti-TIP2 (N-19), rabbit polyclonal anti-E-cadherin (H-108) and mouse monoclonal anti-vimentin (V-9).

    Techniques: Transfection, Luciferase, Expressing, Western Blot

    Loss of hScrib reduces total HPV-18 E6 protein levels. HeLa cells were transfected with siRNA Luciferase or siRNA hScrib and after 72 h the cells were harvested and fractionated into cytosolic (F1), membrane (F2), nuclear (F3) and cytoskeletal (F4) fractions. These were then analysed by western blotting for HPV-18 E6, p53 and hScrib. Also shown are the loading controls for each fraction.

    Journal: Papillomavirus Research

    Article Title: The high-risk HPV E6 target scribble (hScrib) is required for HPV E6 expression in cervical tumour-derived cell lines

    doi: 10.1016/j.pvr.2016.04.001

    Figure Lengend Snippet: Loss of hScrib reduces total HPV-18 E6 protein levels. HeLa cells were transfected with siRNA Luciferase or siRNA hScrib and after 72 h the cells were harvested and fractionated into cytosolic (F1), membrane (F2), nuclear (F3) and cytoskeletal (F4) fractions. These were then analysed by western blotting for HPV-18 E6, p53 and hScrib. Also shown are the loading controls for each fraction.

    Article Snippet: The following antibodies were purchased from Santa Cruz Biotechnology: mouse monoclonal anti-p53 (DO-1), mouse monoclonal anti-α-actinin (H-2), mouse monoclonal anti-Dlg (2D11), goat polyclonal anti-Scribble (C-20), goat polyclonal anti-TIP2 (N-19), rabbit polyclonal anti-E-cadherin (H-108) and mouse monoclonal anti-vimentin (V-9).

    Techniques: Transfection, Luciferase, Western Blot

    Loss of hScrib decreases the levels of HPV-18 E6 mRNA. Panel A. HeLa cells were transfected with siRNA against Luciferase or siRNA HPV-18 E6/E7 or siRNA hScrib. 72 h after transfection, cells were harvested and the expression patterns of: pRB as a surrogate marker for E7 expression, p53 to monitor E6/E7 loss, hScrib and α-actinin, to monitor the protein loading, were assessed by western blot. Panel B. Band intensities of hypo-phosphorylated and hyper-phosphorylated pRB were determined using the OptiQuant quantification program. Levels of pRB expression are expressed as the % of hyper- and hypo-phosphorylated pRB relative to siLuciferase-transfected control cells. Standard deviations are also shown. Panel D. The graph shows the result of quantitative real time pcr for the levels of E6 mRNA, using GAPDH as the control. The bars show the relative levels of E6 expression where siLuc is used as the reference. Note the greater than 60% reduction in E6 mRNA following transfection with either siE6/E7 or siScrib. The numbers represent the means from 3 independent experiments and standard deviations are shown. Panel C shows the accompanying western blot verifying ablation of hScrib expression in a representative assay.

    Journal: Papillomavirus Research

    Article Title: The high-risk HPV E6 target scribble (hScrib) is required for HPV E6 expression in cervical tumour-derived cell lines

    doi: 10.1016/j.pvr.2016.04.001

    Figure Lengend Snippet: Loss of hScrib decreases the levels of HPV-18 E6 mRNA. Panel A. HeLa cells were transfected with siRNA against Luciferase or siRNA HPV-18 E6/E7 or siRNA hScrib. 72 h after transfection, cells were harvested and the expression patterns of: pRB as a surrogate marker for E7 expression, p53 to monitor E6/E7 loss, hScrib and α-actinin, to monitor the protein loading, were assessed by western blot. Panel B. Band intensities of hypo-phosphorylated and hyper-phosphorylated pRB were determined using the OptiQuant quantification program. Levels of pRB expression are expressed as the % of hyper- and hypo-phosphorylated pRB relative to siLuciferase-transfected control cells. Standard deviations are also shown. Panel D. The graph shows the result of quantitative real time pcr for the levels of E6 mRNA, using GAPDH as the control. The bars show the relative levels of E6 expression where siLuc is used as the reference. Note the greater than 60% reduction in E6 mRNA following transfection with either siE6/E7 or siScrib. The numbers represent the means from 3 independent experiments and standard deviations are shown. Panel C shows the accompanying western blot verifying ablation of hScrib expression in a representative assay.

    Article Snippet: The following antibodies were purchased from Santa Cruz Biotechnology: mouse monoclonal anti-p53 (DO-1), mouse monoclonal anti-α-actinin (H-2), mouse monoclonal anti-Dlg (2D11), goat polyclonal anti-Scribble (C-20), goat polyclonal anti-TIP2 (N-19), rabbit polyclonal anti-E-cadherin (H-108) and mouse monoclonal anti-vimentin (V-9).

    Techniques: Transfection, Luciferase, Expressing, Marker, Western Blot, Real-time Polymerase Chain Reaction