Journal: Cell Death and Differentiation

Article Title: ROS-dependent activation of JNK converts p53 into an efficient inhibitor of oncogenes leading to robust apoptosis

doi: 10.1038/cdd.2013.186

Figure Lengend Snippet: ROS-mediated activation of JNK contributes to the p53-mediated apoptosis, DDR and transcriptional repression of oncogenes. ( a ) Dose-dependent induction of p-JNK, p-Ser33-p53, p-Ser15-p53, γ H2AX, PARP cleavage and inhibition of Wip1, Mcl1 and MdmX by RITA as assessed by western blotting. ( b ) HCT116 and HCT116 p53−/− cells were treated with 1 μ M RITA; analyzed as in ( a ). ( c ) Pretreatment with NAC for 6 h prevented the induction of p-JNK, γ H2AX, PARP cleavage and inhibition of Wip1, Mcl1 and MdmX by RITA as analyzed by immunoblotting. ( d ) JNK inhibitor SP600125 prevented the induction of p-JNK, p-Ser33-p53, γ H2AX, PARP cleavage and inhibition of Wip1, Mcl1 and MdmX by RITA, as assessed by western blotting. ( e ) SP600125 blocked the induction of p-JNK, p-Ser33-p53, γ H2AX, PARP cleavage and inhibition of MdmX and Wip1 by combination treatment with 0.05 μ M RITA and 1 μ M Aura for 24 h, as assessed by western blotting.( f , g ) Depletion of JNK by siRNA prevented the induction of γ H2AX ( f ), p-Ser33-p53 and inhibition of Wip1 and MdmX ( g ), analyzed by immunoblotting. ( h ) Inhibition of JNK by siRNA prevented apoptosis induction by RITA, as measured by FACS of PI-stained cells. ( i ) SP600125 blocked the repression of MCL1, PPM1D, PIK3CA, PIK3CB, EIF4E and MDM4 (MdmX) mRNA upon RITA treatment as assessed by qPCR (mean±S.E.M., n =3) in HCT116 (12 h treatment) and MCF7 (8 h treatment) cells

Article Snippet: Plasmids encoding FLAG-Wip1 and shRNA for Wip1 were kindly provided by René H. Medema, Utrecht, Netherlands.

Techniques: Activation Assay, Inhibition, Western Blot, Staining

Journal: Cell Death and Differentiation

Article Title: ROS-dependent activation of JNK converts p53 into an efficient inhibitor of oncogenes leading to robust apoptosis

doi: 10.1038/cdd.2013.186

Figure Lengend Snippet: Inhibition of Wip1 promotes the induction of γ H2AX upon RITA treatment. ( a ) Wip1 mRNA was repressed after 8 h treatment with 1 μ M RITA, but not 0.1 μ M RITA, as assessed by qPCR (mean±S.E.M., n =3). ( b ) Downregulation of Wip1 protein level correlated with the induction of γ H2AX upon RITA treatment as analyzed by immunoblotting. ( c ) MCF7 and U2OS cells stably transfected with empty vector shRNA or Wip1 shRNA were treated with 0.1 and 1 μ M RITA for indicated periods and γ H2AX was assessed as in ( b ). ( d ) HCT116 and U2OS cells transfected with either empty vector or FLAG-Wip1 were treated with 1 μ M RITA for indicated times. Proteins were detected by western blotting

Article Snippet: Plasmids encoding FLAG-Wip1 and shRNA for Wip1 were kindly provided by René H. Medema, Utrecht, Netherlands.

Techniques: Inhibition, Western Blot, Stable Transfection, Transfection, Plasmid Preparation, shRNA

Journal: Cell Death and Differentiation

Article Title: ROS-dependent activation of JNK converts p53 into an efficient inhibitor of oncogenes leading to robust apoptosis

doi: 10.1038/cdd.2013.186

Figure Lengend Snippet: Depletion of Wip1 confers a sustained transcriptional activation of p53 target genes, but does not facilitate transrepression. ( a and b ) Microarray analysis of MCF7 cells with (indicated in violet) or without (indicated in gray) Wip1 depletion by shRNA, treated with 0.1 μ M RITA or DMSO for indicated time points revealed that p53-mediated transactivation was enhanced by Wip1 silencing. ( c ) Wip1 downregulation led to the increased induction of p53-activated genes (upper panel) but did not augment the repression of pro-survival genes by p53 (lower panel) upon low dose of RITA as analyzed by qPCR (mean±S.E.M., n =3). Insert demonstrates the efficiency of Wip1 depletion, as assessed by immunoblotting. ( d ) MCF7 cells transfected with either empty vector or FLAG-Wip1 were treated with 1 μ M RITA or DMSO for 8 h and mRNA levels of PIK3CA , PIK3CB and IGF-1R were assessed by qPCR (mean±S.E.M., n =3). ( e ) MCF7 cells stably transfected with shWip1 or control shVector were treated with 0.1 and 1 μ M RITA or DMSO for 24 h, and cells were stained with Annexin V followed by FACS analysis (mean±S.E.M., n =3, * P <0.05, ** P <0.01, by two-tailed t -test). ( f ) Model of the synthetic lethality upon activation of p53 and inhibition of TrxR. Inhibition of TrxR lead to the accumulation of ROS and activation of JNK, facilitating p53 function upon its release from Mdm2. In turn, activated p53 induces pro-oxidant genes, which increases the level of ROS, further activating JNK, and thus, p53. Activated JNK converts p53 to an inhibitor of Wip1 and MdmX, therefore amplifying p53 activity. Transcriptional repression of Mcl1, eIF4E and PI3K abolishes survival signaling, contributing to apoptosis induction. Thus, the dual targeting of p53 and TrxR (i.e., by RITA) leads to the robust apoptosis

Article Snippet: Plasmids encoding FLAG-Wip1 and shRNA for Wip1 were kindly provided by René H. Medema, Utrecht, Netherlands.

Techniques: Activation Assay, Microarray, shRNA, Western Blot, Transfection, Plasmid Preparation, Stable Transfection, Control, Staining, Two Tailed Test, Inhibition, Activity Assay

Journal:

Article Title: Dissociating the Centrosomal Matrix Protein AKAP450 from Centrioles Impairs Centriole Duplication and Cell Cycle Progression

doi: 10.1091/mbc.E02-09-0614

Figure Lengend Snippet: BrdU incorporation and FACSCAN analysis of human diploid RPE1 fibroblasts in the absence or presence of Myc-C-ter AKAP450 expression (low level)

Article Snippet: Transfection of HeLa Cells HeLa cells and Chinese hamster ovary (CHO) cells were grown in DMEM medium supplemented with 10% fetal calf serum and human RPE1 fibroblasts (BD Biosciences Clontech) were grown in DMEM/F-12 medium supplemented with 10% fetal calf serum.

Techniques: BrdU Incorporation Assay, Expressing