Journal: Cell Death and Differentiation

Article Title: NPD1-mediated stereoselective regulation of BIRC3 expression through cREL is decisive for neural cell survival

doi: 10.1038/cdd.2014.233

Figure Lengend Snippet: Stereospecificity of NPD1 bioactivity selectively upregulates BIRC3 expression. ( a ) BIRC1 to 8 in response to 600 μ M H 2 O 2 /10 ng/ml TNF- α in the presence or absence of 100 nM NPD1 at 2, 4 and 6 h of treatment. ( b ) BIRC3 mRNA expression in response to 100 nM maresin 1, lipoxin-A4 and RvE1 along with NPD1 and its stereoisomers SS-NPD1 and RR-NPD1 and ( c ) in an siRNA dose-dependent curve. ARPE-19 cells were transfected with 0, 5, 10, 20, 50 and 100 pmol of siRNA per ml of culture media and treated with 400 μ M H 2 O 2 /10 ng/ml TNF- α in the presence or absence of 100 nM NPD1. ( i – vi ) Structure of the lipid mediators used in ( b ). The bars represent the mean of three triplicates ± standard error. * P <0.05, NS=non-significant P- value. NPD1-treated samples=blue bars; OS+NPD1=light blue bars

Article Snippet: Wild-type BIRC3-, cREL- and cREL-fused-with-GFP-expression constructs were obtained from Origene.

Techniques: Expressing, Transfection

Journal: Cell Death and Differentiation

Article Title: NPD1-mediated stereoselective regulation of BIRC3 expression through cREL is decisive for neural cell survival

doi: 10.1038/cdd.2014.233

Figure Lengend Snippet: TNFR1 stably-silenced cells display enhanced BIRC3 expression and cell survival upon oxidative stress (OS). ( a and b ) TNFR1 and NC shRNA-expressing ARPE-19 cells were subjected to OS in the presence or absence of NPD1. ( a ) Representative pictures and ( b ) quantification of apoptotic TNFR1 and NC shRNA-expressing cells in the presence or absence of 50 nM NPD1. ( c ) BIRC3 expression induced by NPD1 upon OS by the means of real-time PCR in TNFR1-deficient cells. ( d and e ) Western blot showing the time-dependent phosphorylation of ( d ) I k B α and ( e ) I k B β after 0, 15, 30 and 60 min of OS treatment in the presence or absence of 100 nM NPD1. ( f ) NPD1 effects on canonical NF- κ B activation measured by the means of luciferase reporter assay in OS conditions. OS: 600 μ M H 2 O 2 /10 ng/ml TNF- α . NPD1: 100 nM. Bars represent the mean of triplicates ± standard error of the mean. * P <0.05, NS=non-significant P -value. NPD1 treated samples=blue bars; OS+NPD1=light blue bars

Article Snippet: Wild-type BIRC3-, cREL- and cREL-fused-with-GFP-expression constructs were obtained from Origene.

Techniques: Stable Transfection, Expressing, shRNA, Real-time Polymerase Chain Reaction, Western Blot, Activation Assay, Luciferase, Reporter Assay

Journal: Cell Death and Differentiation

Article Title: NPD1-mediated stereoselective regulation of BIRC3 expression through cREL is decisive for neural cell survival

doi: 10.1038/cdd.2014.233

Figure Lengend Snippet: NPD1-mediated BIRC3 promoter activation. BIRC3 promoter was analyzed using a luciferase reporter assay. ( a ) Schematic representation of the constructs used for deletion and mutation. ( b ) BIRC3 promoter deletion analysis using constructs containing 527, 247, 200, 174 and 93 bp (depicted in a ) upstream of the transcription start site. Transfected cells were treated with 130 μ M H 2 O 2 /10 ng/ml TNF- α in the presence or absence of 200 nM NPD1. ( c ) Site directed mutation analysis on the NF- κ B sites. Cells were treated with 400 μ M H 2 O 2 /10 ng/ml TNF- α in the presence or absence of 200 nM NPD1. ( d ) Luciferase activity was standardized by GFP fluorescence. Bars represent the mean of triplicates ± standard error of the mean. * P <0.05, NS=non-significant P- value. NPD1-treated samples=blue bars; OS+NPD1=light blue bars

Article Snippet: Wild-type BIRC3-, cREL- and cREL-fused-with-GFP-expression constructs were obtained from Origene.

Techniques: Activation Assay, Luciferase, Reporter Assay, Construct, Mutagenesis, Transfection, Activity Assay, Fluorescence

Journal: Cell Death and Differentiation

Article Title: NPD1-mediated stereoselective regulation of BIRC3 expression through cREL is decisive for neural cell survival

doi: 10.1038/cdd.2014.233

Figure Lengend Snippet: cREL nuclear translocation and binding to BIRC3 promoter to induce the activation of its expression. Changes of distribution, activity and expression of cRel were assessed at three time points (2, 4 and 6 h) in ARPE-19 cells treated with 400 μ M H 2 O 2 /10 ng/ml TNF- α in the presence or absence of 100 nM NPD1. ( a – c ) Immunocytochemistry of cREL in cells: ( a ) representative pictures showing the distribution of the cREL signal (red). Nuclei were stained with DAPI (blue). ( b and c ) Portion of cells depicting cREL nuclear or cytoplasmic signal. ( d and e ) cREL protein content evaluated by western blot ( d ) in the nuclear fraction standardized using TBP at 2 h and ( e ) in whole cells standardized by GAPDH after 4 h of OS or OS+NPD1. ( f ) ChiP assay at 4 h showing the binding of cREL to BIRC3 promoter. The co-immunoprecipitated genomic DNA was amplified and standardized by the input. ( g ) Co-immunoprecipitation of cREL and p65/RelA at 4 h of treatment standardized by GAPDH. ( h ) p65/RelA, RelB and cRel expression determined by real-time PCR. ( i – k ) Silencing of cRel induced changes in the expression of: ( i ) cREL, ( j ) RelB and ( k ) BIRC3 in human RPE (hRPE) cells established by the means of real-time PCR. Concentrations of 2.5, 10 and 50 pmol siRNA per ml of cell culture medium were used to show a concentration-dependent effect on the expression at 4 h. ( l and m ) Schematization of the temporal pattern of ( l ) cREL, RelB and BIRC3 expression and ( m ) cREL translocation. The values are represented as the mean of triplicates ± the standard error. * P <0.05, NS=non-significant P- value. NPD1-treated samples=blue bars; OS+NPD1=light blue bars

Article Snippet: Wild-type BIRC3-, cREL- and cREL-fused-with-GFP-expression constructs were obtained from Origene.

Techniques: Translocation Assay, Binding Assay, Activation Assay, Expressing, Activity Assay, Immunocytochemistry, Staining, Western Blot, Immunoprecipitation, Amplification, Real-time Polymerase Chain Reaction, Cell Culture, Concentration Assay

Journal: Cell Death and Differentiation

Article Title: NPD1-mediated stereoselective regulation of BIRC3 expression through cREL is decisive for neural cell survival

doi: 10.1038/cdd.2014.233

Figure Lengend Snippet: NPD1 fails to rescue BIRC3 silenced cells. ( a – e ) Apoptosis noted as percentage of Hoechst- or TUNEL-positive cells was assessed on ARPE-19 ( a – c ) or primary human RPE (hRPE) cells ( d and e ). ( a ) Representative images of TUNEL staining performed on ARPE-19 cells, ( b and c ) Quantification of TUNEL and Hoechst-positive cells when transfected with BIRC3 or control siRNA and treated with 0, 400 and 600 μ M H 2 O 2 /10 ng/ml TNF- α with or without 200 nM NPD1. BIRC3 ( d ), cREL ( e ) or control siRNA-transfected hRPE cells treated with 600 μ M H 2 O 2 /10 ng/ml TNF- α in the presence or absence of NPD1 100 nM. cREL and BIRC3 ( f ) and BIRC3 ( g ) protein content when cREL and BIRC3, respectively, were overexpressed. ( h ) Percentage of apoptosis in ARPE-19 cells overexpressing cREL or BIRC3 when confronted with OS in the presence or absence of NPD1. ( i and j ) Overexpression of ( i ) cREL or ( j ) BIRC3 using a wild-type open reading frame (ORF) or one carrying silent mutations at the siRNAs binding sites (ORFmut) to prevent its silencing and rescue from the knocked down phenotype. Upper panels show representative western blots for the corresponding protein content in each sample. Lower panels depict percentage of apoptosis for each treatment. ( k and l ) Activation of effector caspases 3 and 7 as a result of OS treatment in BIRC3-silenced cells. ( k ) Representative nuclei and staining of cells: upper panel, control; lower panel, OS. ( m and n ) Apoptosis and necrosis measured by the means of AnnexinV and 7-Amino actinomycinD when cells were treated with OS and NPD1 in the presence of z-VAD, a pan caspase inhibitor or Necrostatin 1 (Nec1). ( o ) cREL translocation (upper panel) and percentage of apoptosis of control and cREL-silenced (lower panel) ARPE-19 cells subjected to OS with the addition NPD1 or DHA plus 10 ng/ml of PEDF, FGF, CNTF or BDNF to endogenously induce NPD1 synthesis. ( p ) Schematization of the model obtained by the integration of the data obtained in this report and the context. Bars represent the mean of triplicates + standard error of the mean. * P <0.05 NS=non-significant P -value. NPD1-treated samples=blue bars; OS+NPD1=light blue bars; DHA+growth factors=teal bars; OS+DHA+growth factors=light teal bars

Article Snippet: Wild-type BIRC3-, cREL- and cREL-fused-with-GFP-expression constructs were obtained from Origene.

Techniques: TUNEL Assay, Staining, Transfection, Over Expression, Binding Assay, Western Blot, Activation Assay, Translocation Assay

Journal: Cell Death and Differentiation

Article Title: NPD1-mediated stereoselective regulation of BIRC3 expression through cREL is decisive for neural cell survival

doi: 10.1038/cdd.2014.233

Figure Lengend Snippet: BIRC3 mediates the pro-survival response induced by the DHA/NPD1 pathway in an ischemia-reperfusion stroke model. ( a – c ) Model for inducing ischemia-reperfusion by middle cerebral artery occlusion (MCAo) in rats. ( a ) Timeline showing surgery, treatment and tests performed. ( b ) Coronal brain diagram (bregma level –0.3 mm) showing locations of regions for western blot and immunohistochemistry for , and lipidomic analysis (A: anterior; P: posterior). ( c ) Diagram of MCAo model obtained by introducing intraluminal filament (red). ( d ) Total neurological score (normal score =0, maximal deficit=12), tactile placing (proprioceptive, lateral, dorsal reactions; normal score=0, maximal deficit=2) in rats after MCAo. DHA treatment improved the total and placing deficits on days 1, 3 and 7 compared with the saline-treated group. ( e ) Content of NPD1 and a second product of the stabilized precursor, 17HDHA, in penumbra regions of rats subjected to MCAo and treated with DHA or vehicle as a control. Data are means±standard error of the mean; n =6 per group. * P <0.05 in repeated-measures, ANOVA followed by Bonferroni test. DHA treatment=teal bars

Article Snippet: Wild-type BIRC3-, cREL- and cREL-fused-with-GFP-expression constructs were obtained from Origene.

Techniques: Western Blot, Immunohistochemistry

Journal: Cell Death and Differentiation

Article Title: NPD1-mediated stereoselective regulation of BIRC3 expression through cREL is decisive for neural cell survival

doi: 10.1038/cdd.2014.233

Figure Lengend Snippet: DHA/NPD1 induce translocation of cREL and increased BIRC3 in vivo . ( a ) Western blot on days 1, 3 and 7 after DHA treatment performed on the posterior section. ( b – e ) Translocation of cREL in neurons of the penumbra, areas A1-3 and P1-3 (see ). ( b ) Representative images of nuclear translocation of cREL in saline- and DHA-treated animals in the P2 region 1 day after treatment. (NeuN red; c-REL green). ( c ) High magnification. cREL ( d ) total (upper panels) and ( e ) nuclear (lower panels) quantification from areas A1 to 3 (upper panels) and P1-3 (lower panels). ( f ) BIRC3 (red) and NeuN (green), and ( g ) BIRC3 (red) and GFAP (green) double staining on day 1 after stroke. ( h ) Quantification of the co-localization studies in ( f and g) . Data are means±S.E.M.; n =6 per group. * P <0.05 in repeated-measures, ANOVA followed by Bonferroni test. DHA treatment=teal bars

Article Snippet: Wild-type BIRC3-, cREL- and cREL-fused-with-GFP-expression constructs were obtained from Origene.

Techniques: Translocation Assay, In Vivo, Western Blot, Double Staining