Journal: Journal of Biological Chemistry

Article Title: c-IAP1 and c-IAP2 Are Critical Mediators of Tumor Necrosis Factor α (TNFα)-induced NF-κB Activation

doi: 10.1074/jbc.c800128200

Figure Lengend Snippet: FIGURE1.TNF-stimulatedNF-Bactivationdependsonc-IAP1andc-IAP2.A,analysisofIBproteinlevels in WT and in c-IAP1-, c-IAP2-, and XIAP-deficient MEFs. Cells were treated for the indicated periods of time with 20 ng/ml recombinant murine TNF. The protein levels of IB were analyzed by Western blotting. KO, knock- out. B, c-IAP2-null MEFs were transfected with scrambled (Contr.) or c-IAP1-specific siRNA duplexes. 48 h later, cells were treated with recombinant murine TNF (20 ng/ml), and protein levels were analyzed as in A. C, HT1080 cells werepretreatedwithvehicle(DMSO)orBV6(5M)for5hfollowedbytreatmentwithhumanrecombinantTNF(20 ng/ml) for the indicated time periods. The protein levels of IB and c-IAP1 were analyzed by Western blotting. D, down-regulation of c-IAP1 and c-IAP2 expression reduces viability of TNF-treated cells. Cells were transfected with scrambled, c-IAP1-specific, or c-IAP2-specific siRNA duplexes. 48 h later, cells were treated with TNF as indi- cated. Cell viability was determined as described under “Experimental Procedures,” and protein levels were deter- mined as described as in A. The error bars represent standard deviation from three independent experiments. The insetshowsdown-regulationofc-IAP2inc-IAP1-deficientMEFs;down-regulationofc-IAP1inc-IAP2-deficientMEFs is presented in panel B. Western blotting was performed with indicated antibodies.

Article Snippet: The primary antibodies against mouse c-IAP1 were kindly provided by Drs. John Silke and David Vaux; anti-human c-IAP1 antibodies were purchased from R&D (affinity-purified goat antibody) or Protein Tech Group Inc.; pan c-IAP1/2 human/murine antibody was from R&D; and anti-c-IAP2 antibodies were purchased from AbCam.

Techniques: Recombinant, Western Blot, Knock-Out, Transfection, Expressing, Standard Deviation

Journal: Journal of Biological Chemistry

Article Title: c-IAP1 and c-IAP2 Are Critical Mediators of Tumor Necrosis Factor α (TNFα)-induced NF-κB Activation

doi: 10.1074/jbc.c800128200

Figure Lengend Snippet: FIGURE 2. c-IAPs are critical for TNF-induced RIP1 polyubiquitination. A, HT1080 cells were pre- treated with DMSO or BV6 (5 M) for 5 h followed by TNF treatment as indicated. Cell lysates were immunoprecipitated (IP) with anti-TNFR1 antibodies, and protein levels in cellular lysates and in the TNFR1-associated complex were determined by Western blotting with the indicated antibodies. Ub, ubiq- uitin. B, WT or c-IAP1-null MEF cells were treated for the indicated periods of time with FLAG-tagged TNF (1 mg/ml) followed by immunoprecipitation and Western blot analysis with the indicated antibodies. One-half of the immunoprecipitated protein complexes described above were dissociated by a 20-min incubation in 6 M urea. Collected supernatants were diluted 25-fold in the lysate buffer followed by immunoprecipitation and Western blot analysis with the indicated antibodies. KO, knock-out. C, RIP1 was immunoprecipitated from c-IAP1-null MEFs and incubated for 45 min in ubiquitination reactions with the indicated combination of recombinant c-IAP1, E2 enzymes, and ubiquitin proteins (WT, Lys-48 only, or Lys-63 only). RIP1 modifications were determined with anti-RIP1 antibodies. D, recombinant RIP1 was incubated for 45 min in ubiquitination reaction in the absence or presence of recombinant c-IAP1 or c-IAP1 RING mutant (Rm; H588A) and the indicated combination of E2 enzymes and ubiquitin proteins (WT, Lys-48 only, or Lys-63 only). RIP1 modifications were determined with anti-RIP1 antibodies.

Article Snippet: The primary antibodies against mouse c-IAP1 were kindly provided by Drs. John Silke and David Vaux; anti-human c-IAP1 antibodies were purchased from R&D (affinity-purified goat antibody) or Protein Tech Group Inc.; pan c-IAP1/2 human/murine antibody was from R&D; and anti-c-IAP2 antibodies were purchased from AbCam.

Techniques: Immunoprecipitation, Western Blot, Incubation, Knock-Out, Ubiquitin Proteomics, Recombinant, Mutagenesis

Journal: Cell Death and Differentiation

Article Title: cIAP1 regulates the EGFR/Snai2 axis in triple-negative breast cancer cells

doi: 10.1038/s41418-018-0100-0

Figure Lengend Snippet: Treatment with SM83 reduces the expression of Snai2 both in vivo and in vitro. a NOD/SCID mice engrafted subcutaneously with 5 × 10 6 MDA-MB231 were treated with intraperitoneal injection of SM83 (5 mg/kg, 5 times/week) or left untreated (4 mice/group) until the end of the experiment. Schedule of the experiment (upper panel) and tumor volumes (bottom panel) measured twice a week (significant differences in days 24, 27, and 30. * P = 0.0476, 0.0391, and 0.0344, respectively. Unpaired two-tailed t- test). b Six hours after the last injection, mice were killed, nodules collected, and analyzed by western blot to detect the levels of SM83 targets cIAP1, cIAP2, and XIAP. Actin and vinculin are shown as loading controls. c MDA-MB231 cells transfected in vitro with two siRNAs targeting cIAP1 were treated or not with 100 nM SM83 for 1 h. Western blots were performed to evaluate the levels of cIAP1, cIAP2, and XIAP 72 h after transfection. Values show the fold levels of XIAP. d Differentially expressed genes: heat map showing the 50 genes significantly upregulated and the 15 downregulated by SM83 in MDA-MB231 nodules collected as described in Fig. 1a. e Wound-healing experiments performed with MDA-MB231 cells transfected with control (NT1) or Snai2-specific siRNAs ( n = 4, ** P = 0.0033. Unpaired two-tailed t- test). The graph shows the percentage of gap closure after 24 h of migration. The complete experiment with the other siRNAs tested is shown in Fig. S . f The levels of Snai2, downregulated in the GEP shown in Fig. 1d, and LRIG1, upregulated, were evaluated by western blot performed with lysates of MDA-MB231 nodules. Values show the fold levels of Snai2 and LRIG1 normalized to Actin levels. g Levels of Snai2 in MDA-MB231 cells treated with 100 nM SM83 in time-course experiments. Cleavage of p100 NF-kB2 into the p52 form was used to verify the expected activation of the non-canonical NF-kB pathway upon SM83 administration

Article Snippet: Membranes were then saturated for 30 min in Tris-buffered saline containing 4% BSA and incubated overnight with the anti-human primary antibodies recognizing cIAP1 #ab108361, total EGFR #ab30 (Abcam); Snai2 #9585, LRIG1 #12752, NF-kB2 #4882, NF-kB1 #3035, Cleaved Caspase3 #9501, p65/RelA #3034, pEGFR Tyr1068 #2236, c-Cbl #2747, Myc-Tag #2278 (Cell Signaling Technology, Danvers, MA, USA); phospho-ERK1/2 #M8159, ERK1/2 #M5670, Actin #A1978 and Vinculin #V9131 (Sigma-Aldrich), cIAP2 #552783 and XIAP #610763 (BD Biosciences).

Techniques: Expressing, In Vivo, In Vitro, Injection, Two Tailed Test, Western Blot, Transfection, Control, Migration, Activation Assay

Journal: Cell Death and Differentiation

Article Title: cIAP1 regulates the EGFR/Snai2 axis in triple-negative breast cancer cells

doi: 10.1038/s41418-018-0100-0

Figure Lengend Snippet: Snai2 expression is promoted by cIAP1, but not cIAP2 or XIAP. a Western blot showing the levels of Snai2 in MDA-MB231 cells transfected with siRNAs specific for cIAP1 or cIAP2, and, after 72 h, treated with 100 nM SM83 for further 6 h. b MDA-MB231 and BT549 cells were transfected as in Fig. 2a with siRNAs targeting XIAP and cIAP1, and western blot was performed to detect the levels of Snai2. c MDA-MB231, BT549, SUM159, and MDA-MB157 cells were transfected with two siRNAs specific for cIAP1 and the levels of Snai2 were detected. d The same cell lines employed in Fig. 2c were treated with 100 nM SM83 and viability tested with CellTiter-Glo assay 24 h later (two-tailed paired t- test, n = 3. MDA-MB231 ** P = 0.0011; BT549 ** P = 0.0082; SUM159 and MDA-MB157; ns not significant). e SM83 (100 nM) was used to treat MDA-MB231 and BT549 cells, pre-treated or not 1 h with 20 µM z-VAD, for 6 h and then cells were analyzed by western blot to detect the total levels of Snai2 and cIAP1, and the cleaved form of caspase-3. Values show the fold levels of Snai2 relative to untreated cells

Article Snippet: Membranes were then saturated for 30 min in Tris-buffered saline containing 4% BSA and incubated overnight with the anti-human primary antibodies recognizing cIAP1 #ab108361, total EGFR #ab30 (Abcam); Snai2 #9585, LRIG1 #12752, NF-kB2 #4882, NF-kB1 #3035, Cleaved Caspase3 #9501, p65/RelA #3034, pEGFR Tyr1068 #2236, c-Cbl #2747, Myc-Tag #2278 (Cell Signaling Technology, Danvers, MA, USA); phospho-ERK1/2 #M8159, ERK1/2 #M5670, Actin #A1978 and Vinculin #V9131 (Sigma-Aldrich), cIAP2 #552783 and XIAP #610763 (BD Biosciences).

Techniques: Expressing, Western Blot, Transfection, Glo Assay, Two Tailed Test

Journal: Cell Death and Differentiation

Article Title: cIAP1 regulates the EGFR/Snai2 axis in triple-negative breast cancer cells

doi: 10.1038/s41418-018-0100-0

Figure Lengend Snippet: EGFR promotes Snai2 expression in a MAPK-dependent manner. a MDA-MB231, BT549, and MDA-MB157 cells were harvested after treatment for 2 h with 10 μM inhibitor of PI3K (LY294002), AKT (Triciribine), MEK (UO126), and p38 (SB203580). Western blot was performed to analyze the levels of Snai2. b MDA-MB231 cells were transfected with siRNAs specific for cIAP1, ERK1, and ERK2 to detect the levels of Snai2 72 h after transfection. cIAP1 and ERK1/2 are shown to control the silencing efficiency. c A panel of breast cancer cell lines was tested to compare the levels of Snai2. BaA basal “A”, BaB basal “B”, Lu Luminal . d For each cancer type available in the TCGA study, Spearman’s correlation between EGFR and SNAI2 was calculated using RNA-Seq data (expressed as log2 counts per million mapped reads). Only primary tumors were considered in the analysis. Red arrow indicates the correlation bar in breast cancers. e BT549 cells were serum-starved overnight and then stimulated with 20 ng/ml EGF and TGFα in time-course experiments. Snai2 levels are shown together with total and activated levels of EGFR. MDA-MB231 ( f ) and BT549 ( g ) cells were serum-starved overnight, pre-treated or not with 100 μg/ml cetuximab for 1 h and then stimulated with 20 ng/ml EGF for the indicated time points. Western blot was performed to detect Snai2 levels, total ERK1/2 and EGFR, and their activated levels. Values show the fold levels of Snai2 relative to untreated cells. h Human mammary epithelial cell lines, parental and bearing mutated EGFR, were serum-starved and stimulated with 20 ng/ml EGF for the indicated times to evaluate Snai2 levels

Article Snippet: Membranes were then saturated for 30 min in Tris-buffered saline containing 4% BSA and incubated overnight with the anti-human primary antibodies recognizing cIAP1 #ab108361, total EGFR #ab30 (Abcam); Snai2 #9585, LRIG1 #12752, NF-kB2 #4882, NF-kB1 #3035, Cleaved Caspase3 #9501, p65/RelA #3034, pEGFR Tyr1068 #2236, c-Cbl #2747, Myc-Tag #2278 (Cell Signaling Technology, Danvers, MA, USA); phospho-ERK1/2 #M8159, ERK1/2 #M5670, Actin #A1978 and Vinculin #V9131 (Sigma-Aldrich), cIAP2 #552783 and XIAP #610763 (BD Biosciences).

Techniques: Expressing, Western Blot, Transfection, Control, RNA Sequencing

Journal: Cell Death and Differentiation

Article Title: cIAP1 regulates the EGFR/Snai2 axis in triple-negative breast cancer cells

doi: 10.1038/s41418-018-0100-0

Figure Lengend Snippet: Depletion of cIAP1 hinders EGFR-dependent expression of Snai2. a MDA-MB231 and b BT549 cells were transfected with control or cIAP1-specific siRNAs and, after 48 h, serum-starved overnight. Then, cells were stimulated for the indicated time points with 50 ng/ml and 20 ng/ml EGF, respectively. Levels of Snai2 and activated ERK1/2 were detected, together with cIAP1, to check the transfection efficiency. c BT549 and d MCF10A—wild-type or bearing mutated EGFR—cells were transfected as in Fig. 4a and stimulated with the indicated EGFR ligands (20 ng/ml) to evaluate the expression of Snai2 by western blot. e BT549 and f MCF10A cells were transfected and serum-starved as described before, stimulated 3 h with 20 ng/ml EGF and lysed to extract RNA. Real-time PCR was performed to evaluate Snai2 fold expression relative to GAPDH. BT549: * P = 0.0151, ** P = 0.0036; n = 3; MCF10A: unstimulated siCtr vs. sicIAP1 * P = 0.0290, EGF 3 h siCtr vs. sicIAP1 * P = 0.0330; n = 5; two-tailed paired t- test

Article Snippet: Membranes were then saturated for 30 min in Tris-buffered saline containing 4% BSA and incubated overnight with the anti-human primary antibodies recognizing cIAP1 #ab108361, total EGFR #ab30 (Abcam); Snai2 #9585, LRIG1 #12752, NF-kB2 #4882, NF-kB1 #3035, Cleaved Caspase3 #9501, p65/RelA #3034, pEGFR Tyr1068 #2236, c-Cbl #2747, Myc-Tag #2278 (Cell Signaling Technology, Danvers, MA, USA); phospho-ERK1/2 #M8159, ERK1/2 #M5670, Actin #A1978 and Vinculin #V9131 (Sigma-Aldrich), cIAP2 #552783 and XIAP #610763 (BD Biosciences).

Techniques: Expressing, Transfection, Control, Western Blot, Real-time Polymerase Chain Reaction, Two Tailed Test

Journal: Cell Death and Differentiation

Article Title: cIAP1 regulates the EGFR/Snai2 axis in triple-negative breast cancer cells

doi: 10.1038/s41418-018-0100-0

Figure Lengend Snippet: Silencing of cIAP1 reduces EGFR levels. a BT549 and b MCF10A cells were transfected with control or cIAP1-specific siRNAs before being serum-starved overnight, stimulated with 20 ng/ml EGF and analyzed by western blot to detect the indicate proteins. c BT549 cells were transfected with control or cIAP1-specific siRNAs and, after 48 h, cells were serum-starved for 24 h and then stimulated 30 min with 20 ng/ml EGF. Fixed cells were incubated with anti-EGFR antibody and nuclei stained with DAPI. d LRIG1 expression levels were evaluated by real-time PCR in BT549 cells serum-starved and stimulated with 20 ng/ml EGF after transfection with control or cIAP1-directed siRNAs. Unstimulated siCtr vs. sicIAP1 * P = 0.0175, EGF 3 h siCtr vs. sicIAP1 * P = 0.0341; n = 3; two-tailed paired t- test. e Levels of Snai2 and LRIG1 in BT549 cells silenced for cIAP1 and stimulated or not with 20 ng/ml EGF. f EGFR levels in BT549 cells silenced for cIAP1 and LRIG1, stimulated or not with 20 ng/ml EGF. g LRIG1 expression levels were evaluated by real-time PCR in MCF10A cells serum-starved and stimulated with 20 ng/ml EGF after transfection with control or cIAP1-directed siRNAs. ns not significant; n = 4; two-tailed paired t- test

Article Snippet: Membranes were then saturated for 30 min in Tris-buffered saline containing 4% BSA and incubated overnight with the anti-human primary antibodies recognizing cIAP1 #ab108361, total EGFR #ab30 (Abcam); Snai2 #9585, LRIG1 #12752, NF-kB2 #4882, NF-kB1 #3035, Cleaved Caspase3 #9501, p65/RelA #3034, pEGFR Tyr1068 #2236, c-Cbl #2747, Myc-Tag #2278 (Cell Signaling Technology, Danvers, MA, USA); phospho-ERK1/2 #M8159, ERK1/2 #M5670, Actin #A1978 and Vinculin #V9131 (Sigma-Aldrich), cIAP2 #552783 and XIAP #610763 (BD Biosciences).

Techniques: Transfection, Control, Western Blot, Incubation, Staining, Expressing, Real-time Polymerase Chain Reaction, Two Tailed Test

Journal: Cell Death and Differentiation

Article Title: cIAP1 regulates the EGFR/Snai2 axis in triple-negative breast cancer cells

doi: 10.1038/s41418-018-0100-0

Figure Lengend Snippet: cIAP1 reduces EGFR stability, but promotes its gene transcription. a EGFR and cIAP1 interaction was tested in BT549 cells using PLA assay. b BT549 cells stably expressing Myc/Flag-tagged EGFR were serum-starved and stimulated with 20 ng/ml EGF for the indicated times. Cells were lysed and EGFR was immunoprecipitated with anti-Flag antibody. Western blot was performed to evaluate the interaction of ectopic EGFR with cIAP1 and c-Cbl. c BT549 and MCF10A cells stably expressing Myc/Flag-tagged EGFR were serum-starved, pre-treated with 100 (BT549) or 50 (MCF10A) µg/ml cycloheximide for 30 min and stimulated with 20 ng/ml EGF for the indicated times. Levels of ectopic EGFR were detected with anti-Myc antibody. d Ectopic EGFR was immunoprecipitated as described in Fig. 6b from BT549 cells transfected with control and cIAP1-specific siRNAs. Western blot shows total levels of c-Cbl and the amount of c-Cbl interacting with EGFR. e BT549 and f MCF10A cell stably expressing ectopic Myc/Flag-tagged EGFR were further transduced with lentiviral particles to overexpress c-Cbl or GFP as a control. Cells were silenced for cIAP1, serum-starved, and stimulated with 20 ng/ml EGF as described above, and analyzed by western blot to evaluate the levels of ectopic EGFR by using a Myc-tagged-specific antibody. g BT549 (left panel) and MCF10A (right panel) were silenced for cIAP1 and analyzed by real-time PCR to quantify the levels of EGFR expression fold relative to GAPDH. BT549: unstimulated siCtr vs. sicIAP1 * P = 0.0134, EGF 3 h siCtr vs. sicIAP1 * P = 0.0270; n = 3. MCF10A: unstimulated siCtr vs. sicIAP1 *** P = 0.0004, EGF 3 h siCtr vs. sicIAP1 ** P = 0.0183; n = 4; two-tailed paired t- test

Article Snippet: Membranes were then saturated for 30 min in Tris-buffered saline containing 4% BSA and incubated overnight with the anti-human primary antibodies recognizing cIAP1 #ab108361, total EGFR #ab30 (Abcam); Snai2 #9585, LRIG1 #12752, NF-kB2 #4882, NF-kB1 #3035, Cleaved Caspase3 #9501, p65/RelA #3034, pEGFR Tyr1068 #2236, c-Cbl #2747, Myc-Tag #2278 (Cell Signaling Technology, Danvers, MA, USA); phospho-ERK1/2 #M8159, ERK1/2 #M5670, Actin #A1978 and Vinculin #V9131 (Sigma-Aldrich), cIAP2 #552783 and XIAP #610763 (BD Biosciences).

Techniques: Stable Transfection, Expressing, Immunoprecipitation, Western Blot, Transfection, Control, Transduction, Real-time Polymerase Chain Reaction, Two Tailed Test

Journal: Cell Death and Differentiation

Article Title: cIAP1 regulates the EGFR/Snai2 axis in triple-negative breast cancer cells

doi: 10.1038/s41418-018-0100-0

Figure Lengend Snippet: IAP inhibition hinders EGFR signaling independently from the receptor downregulation. a SUM159 and b MCF10A cells were serum-starved, pre-treated or not with SM83, and stimulated with 20 ng/ml EGF for the indicated times. Western blots were performed to evaluate the total and activated levels of EGFR and ERK1/2, and total levels of Snai2. cIAP1 is shown to control the efficiency of the treatment. c MCF10A cell viability was tested by CellTiter-Glo 24 h after treatment with 100 nM SM83. d BT549 cells ectopically expressing Myc/Flag-tagged EGFR, silenced with control and cIAP1-specific siRNAs, were serum-starved overnight and then stimulated with 20 ng/ml EGF. Western blot was performed to detect the total levels of ectopic EGFR (Myc), cIAP1, and Snai2. e BT549 cells ectopically expressing Myc/Flag-tagged EGFR were serum-starved, pre-treated or not 1 h with SM83, and stimulated with 20 ng/ml EGF for the indicated times. Western blot was performed to detect the total levels of ectopic EGFR (Myc), ERK1/2, cIAP1, and Snai2, and the activated form of ERK1/2. f Tumors described in Fig. were analyzed by western blot to detect the activation of ERK1/2, and the total levels of EGFR and ERK1/2

Article Snippet: Membranes were then saturated for 30 min in Tris-buffered saline containing 4% BSA and incubated overnight with the anti-human primary antibodies recognizing cIAP1 #ab108361, total EGFR #ab30 (Abcam); Snai2 #9585, LRIG1 #12752, NF-kB2 #4882, NF-kB1 #3035, Cleaved Caspase3 #9501, p65/RelA #3034, pEGFR Tyr1068 #2236, c-Cbl #2747, Myc-Tag #2278 (Cell Signaling Technology, Danvers, MA, USA); phospho-ERK1/2 #M8159, ERK1/2 #M5670, Actin #A1978 and Vinculin #V9131 (Sigma-Aldrich), cIAP2 #552783 and XIAP #610763 (BD Biosciences).

Techniques: Inhibition, Western Blot, Control, Expressing, Activation Assay

Journal: Cell Death and Differentiation

Article Title: cIAP1 regulates the EGFR/Snai2 axis in triple-negative breast cancer cells

doi: 10.1038/s41418-018-0100-0

Figure Lengend Snippet: SM83 treatment results in anti-tumor and anti-metastasis effect. a Lungs of NOD/SCID mice bearing MDA-MB231 tumors were collected 2 weeks after the last injection with SM83 (upper panel, see Fig. S for primary tumor volumes), formalin-fixed and paraffin-embedded, and stained with an anti-human vimentin antibody to detect spontaneous metastasis (bottom panel). b Number (untreated n = 7, SM83-treated mice n = 8; sum of two independent experiments shown in Fig. S ; * P = 0.0238. Unpaired two-tailed t- test) and c size (35 metastases/group; * P = 0.0107. Unpaired two-tailed t- test) of spontaneous MDA-MB231 lung metastases were evaluated. d Western blot showing the levels of cIAP1, EGFR, ERK1/2, pERK1/2, and Snai2 in primary tumors at the end of the experiment described in Fig. 8a

Article Snippet: Membranes were then saturated for 30 min in Tris-buffered saline containing 4% BSA and incubated overnight with the anti-human primary antibodies recognizing cIAP1 #ab108361, total EGFR #ab30 (Abcam); Snai2 #9585, LRIG1 #12752, NF-kB2 #4882, NF-kB1 #3035, Cleaved Caspase3 #9501, p65/RelA #3034, pEGFR Tyr1068 #2236, c-Cbl #2747, Myc-Tag #2278 (Cell Signaling Technology, Danvers, MA, USA); phospho-ERK1/2 #M8159, ERK1/2 #M5670, Actin #A1978 and Vinculin #V9131 (Sigma-Aldrich), cIAP2 #552783 and XIAP #610763 (BD Biosciences).

Techniques: Injection, Staining, Two Tailed Test, Western Blot

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