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: International Journal of Molecular Sciences

Article Title: BBS Proteins Affect Ciliogenesis and Are Essential for Hedgehog Signaling, but Not for Formation of iPSC-Derived RPE-65 Expressing RPE-Like Cells

doi: 10.3390/ijms22031345

Figure Lengend Snippet: Investigation of the primary cilium in fibroblast obtained from patients with bardet biedl syndrome (BBS) and in the hTERT-immortalized retinal pigment epithelial cell-line, (RPE-1) transfected with small interfering RNA (siRNA) against the BBS genes. ( A ) IFM analysis of primary cilia. Primary cilia were labeled with anti-ARL13B antibody (green). Nuclei were visualized with DAPI staining (blue). Controls (Ctrl, here CtrlD) are also shown. Scale bars 10 µM. ( B ) Percentage of patient fibroblasts with cilia. Compared to percentage of ciliated cells in a pool of control fibroblasts (Ctrl.pool = CtrL.A + Ctrl.D + Ctrl.E; 92.60%, n = 798 cells), the percentage of ciliated cells was significantly lower in BBS5 −/− (B) (79.54%, n = 308 cells, *** p =5.13 × 10 −10 ), BBS10 −/− (A) (89.16%, n = 369 cells, * p = 0.0494) and BBS10 −/− (B) (85.78%, n = 225 cells, ** p = 0.0015) fibroblasts. No significant differences were observed for BBS1 −/− (94.98%, n = 239, p = 0.202) and BBS5 −/− (A) (92.77%, n = 249, p = 0.9301) compared to the pool of control fibroblasts. There was a significant difference in percentage of ciliated cells between the three controls (Ctrl). The percentage was significantly lower in Ctrl.D (89.18%, n = 305 cells) compared with Ctrl.A (95.72%, n = 234 cells; Ctrl.D/Ctrl.A: * p = 0.0054) and Ctrl.E (93.82%, n = 259 cells; Ctrl.D/Ctrl.E: * p = 0.037). No significant difference was observed between CtrlA and CtrlE (CtrlA/CtrlE: p = 0.34). ( C ) Percentage of siBBS RNA-treated RPE1 cells with cilia. The cilia were labeled with anti ARL13B antibody and nuclei were visualized with DAPI staining. For siRNA efficacy, see . Compared to percentage of ciliated cells in siSCR transfected RPE1 cells (86.81%, n = 379 cells), the percentage of ciliated cells were significantly decreased in RPE1 cells transfected with siBBS5 (60.20%, n = 294 cells, *** p = 2.2802 × 10 −15 ) and siBBS10 (76.40%, n = 322 cells, *** p = 0.000347). No significant differences compared to siSCR transfected cells were obtained for siBBS1 (84.49%, n = 361 cells, p = 0.36789). ( D ) Quantification of primary cilia length in patient fibroblasts. No significant difference was obtained by comparing the three control fibroblasts lines (Ctrl.A, n = 224 cells; Ctrl.D, n = 270 cells; Ctrl.E n = 243 cells. Ctrl.A/Ctrl.D: p = 0.0918; Ctrl.A/Ctrl.E: p = 0.675; Ctrl.D/Ctrl.E: p = 0.207). A pool of all the controls ( n = 737 cells) was used for comparison with the five BBS patient fibroblasts lines. Compared to the controls, BBS1 −/− ( n = 222, p = 3.77 × 10 −11 ) had significantly shorter cilia, whereas BBS5 −/− (A) ( n = 228, *** p = 1.83 × 10 −13 ), BBS5 −/− (B) ( n = 242, *** p = 8.77 × 10 −8 ), BBS10 −/− (A) ( n = 333, *** p = 2.00×10 −16 ) and BBS10 −/− (B) ( n = 202, * p = 0.0165) all had significantly longer cilia. ( E ) Cilia length variation visualized as a boxplot of (C) showing the length variation for all cell-lines. Compared to control cell-lines, BBS5 −/− (A), BBS5 −/− (B) and BBS10 −/− (A) had broader length distributions. ( F ) Quantification of primary cilia length in siBBS-treated RPE1 cells. The cilia length was significant shorter in RPE1 cells treated with siBBS1 ( n = 361, *** p = 2.29 × 10 −8 ), and siBBS10 ( n = 322, *** p = 3.54 × 10 −19 ) compared to siSCR-treated control RPE cells, whereas no significant difference was observed between siBBS5- ( n = 294, p = 0.21) and siSCR-treated cells ( n = 329). SiBBS transfection led to a substantial reduction in expression of the BBS genes . In ( B , C ), the number of cells ( n ) investigated consist of pooled data from three separate experiments and significance was determined using the χ 2 -test. In ( D , F ), the number of cilia ( n ) measured consist of pooled data from three separate experiments. p -values and significance were determined using Student’s t -test, two-tailed. Significance levels p < 0.05, * p < 0.05, and *** p < 0.0005 were used. Error bars represent the standard deviation.

Article Snippet: RPE1 (hTERT-RPE1) cells were a kind gift from Lotte Bang Pedersen (University of Copenhagen, Department of Biology, Section for Cell Biology and Physiology), originally obtained from ATCC (hTERT RPE-1 ATCC ® CRL-4000TM).

Techniques: Transfection, Small Interfering RNA, Labeling, Staining, Control, Comparison, Expressing, Two Tailed Test, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: BBS Proteins Affect Ciliogenesis and Are Essential for Hedgehog Signaling, but Not for Formation of iPSC-Derived RPE-65 Expressing RPE-Like Cells

doi: 10.3390/ijms22031345

Figure Lengend Snippet: Investigation of Hedgehog (Hh) signaling in control and BBS-defective cells. ( A ) Investigation of Hh signaling fibroblast. Expression profile of GLI1 mRNA was normalized to the amount of endogenous GADPH mRNA. Pur-induced expression of GLI1 was significantly increased in control cells compared to patient fibroblasts (BBS1 −/− : * p = 0.015711, BBS5A −/− : * p = 0.012758, BBS5B −/− : * p = 0.014811, BBS10A −/− : * p = 0.014944, BBS10B −/− : * p = 0.013701). Compared to unstimulated cells, a significant increase in GLI1 expression as an effect of pur stimulation was observed in the control (Ctrl), BBS1 −/− and BBS10A −/− fibroblasts (Ctrl: ** p = 0.001787, BBS1 −/− : * p = 0.047977, BBS10A −/− = * p = 0.039275) but not in the other cell types (BBS5A −/− : p = 0.148291, BBS5B −/− : p = 0.232452, BBS10B −/− : p = 0.098018). ( B ) Investigation of Hh signaling in siRNA transfected RPE1 cells. Expression profile of GLI1 was normalized to the amount of endogenous GADPH . Cells transfected with siSCR showed a significant increase in GLI1 expression after pur stimulation (siSCR * p = 0.038869). The increase in siBBS1 and siBBS10 transfected cells was smaller, but still significant, compared to unstimulated cells (siBBS1: * p = 0.020942, siBBS10: * p = 0.046011). No significant effect was observed in siBBS5 transfected cells (siBBS5: p = 0.323142). SiRNA transfection led to a substantial reduction in expression of the BBS genes . ( C ) Ciliary localization of SMO in fibroblast lines. The cells were labeled with anti-AC-TUB (cilia marker, red) and anti-SMO antibody (green). Nuclei were visualized with DAPI staining (blue). Scale bars 10 µM. Arrows indicate ciliary localization. In contrast to control fibroblasts, SMO was observed in a large number of cilia in all the BBS patient fibroblasts. In the control cells, SMO was only observed in a large number of cilia after pur stimulation . ( D ) Quantification of cilia with SMO. The fibroblasts were grown under serum-reduced conditions (0.05% FCS) for 48 h in the presence or absence of 5 µM pur for the final 24 h, as indicated. SMO was present in a large number of the cilia in all BBS patient fibroblasts, both in the presence and the absence of pur. Significantly more SMO was present in the BBS fibroblast cells compared to a pool of control cells (Ctrl.pool: Ctrl.A + Ctrl.D, n = 315) in the absence of pur (BBS1 −/− : *** p = 1.28 × 10 −12 , n = 156, BBS5A −/− : *** p = 2.76 × 10 −8 , n = 145, BBS5B −/− : *** p = 6.95 × 10 −5 , n = 136, BBS10A −/− : *** p = 2.26 × 10 −5 , n = 134, and BBS10B −/− : *** p = 0.00014, n = 141). The control cells had a significant increase in ciliary SMO localization after pur stimulation (*** p = 4.58 × 10 −9 , n = 349/426). All BBS fibroblasts showed a significantly higher basal level of ciliary SMO that only increased significantly in BBS10A after pur stimulation (BBS1 −/− : p = 0.960, n = 222, BBS5A −/− : p = 0.371, n = 228, BBS5B −/− : p = 0.802, n = 242, BBS10A −/− : * p = 0.045, n = 333, BBS10B −/− : p = 0.219, n = 202). All included data were pooled from three independent experiments ( n = 3). In ( A , B ), Student’s t -test, one-tailed, with p < 0.05 significance level was performed. In ( D ), the χ 2 -test was performed with significance levels * p < 0.05, and *** p < 0.0005. Error bars represent the standard deviation.

Article Snippet: RPE1 (hTERT-RPE1) cells were a kind gift from Lotte Bang Pedersen (University of Copenhagen, Department of Biology, Section for Cell Biology and Physiology), originally obtained from ATCC (hTERT RPE-1 ATCC ® CRL-4000TM).

Techniques: Control, Expressing, Transfection, Labeling, Marker, Staining, One-tailed Test, Standard Deviation