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: Oncotarget

Article Title: The BIRC6 gene as a novel target for therapy of prostate cancer: dual targeting of inhibitors of apoptosis

doi:

Figure Lengend Snippet: (A) Correlation of immunohistochemical staining intensity of BIRC6 and clinical (T) stages of prostate cancer (mean staining intensity ± S.E.M.). (B-D) Correlation of BIRC6 immunohistochemical staining intensity with the absence and presence of poor prognostic factors, such as recurrence of PSA, lymph node metastasis and prostatic capsule invasion. The statistical significance of positive trends was determined by the Chi square test for trend. (E) Representative images of correlated expressions between BIRC6 and survivin, XIAP and cIAP1. 20x magnification, scale bar, 100 μm.

Article Snippet: Immunohistochemical staining using rabbit polyclonal antibody against BIRC6 (NB110-40730, Novus Biological, 1:50), rabbit monoclonal antibody against Survivin (#2808, Cell Signaling, 1:50), monoclonal antibody against cIAP1 (MAB8181, R & D Systems, 1:200) and rabbit polyclonal antibody against XIAP (#sc-11426, Santa Cruz, 1:25) was conducted using a Ventana autostainer (model Discover XT; Ventana Medical System, Tucson, AZ) with an enzyme-labelled biotin-streptavidin system and a solvent-resistant DAB Map kit (Ventana).

Techniques: Immunohistochemical staining, Staining

Journal: Oncotarget

Article Title: The BIRC6 gene as a novel target for therapy of prostate cancer: dual targeting of inhibitors of apoptosis

doi:

Figure Lengend Snippet: (A-B) Western blotting showing protein levels of BIRC6, cIAP1 and survivin in two CRPC cell lines (A) PC-3 cells and (B) C4-2 cells transfected with Mock or increasing dosages of scrambled ASO (Scrb), dASOs 6w2 and 6w5 for 72 hr. (C) Comparison of dual IAP targeting and single IAP-targeting. Cell viability of PC-3 cells transfected with dASOs 6w2, 6w5 and siRNA-targeting BIRC6, cIAP1 or survivin, was determined by MTS assay at 72 hr after transfection. Cell viabilities of ASO- and siRNA-treated cells were normalized with corresponding Scrb ASO and siRNA controls. Error bars represent mean percentage cell viability ± S.D. Western blotting of 3 IAPs showing comparable amounts of reduced protein expression obtained with dASO and siRNA single IAP-targeting. (D) Proliferation of PC-3 cells transfected with mock, Scrb ASO, dASOs 6w2 and 6w5. Error bars represent mean cell number ± S.D. (E) MTS viability assay of C4-2 cells treated with dASOs.

Article Snippet: Immunohistochemical staining using rabbit polyclonal antibody against BIRC6 (NB110-40730, Novus Biological, 1:50), rabbit monoclonal antibody against Survivin (#2808, Cell Signaling, 1:50), monoclonal antibody against cIAP1 (MAB8181, R & D Systems, 1:200) and rabbit polyclonal antibody against XIAP (#sc-11426, Santa Cruz, 1:25) was conducted using a Ventana autostainer (model Discover XT; Ventana Medical System, Tucson, AZ) with an enzyme-labelled biotin-streptavidin system and a solvent-resistant DAB Map kit (Ventana).

Techniques: Western Blot, Transfection, Comparison, MTS Assay, Expressing, Viability 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: 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, 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, RNA Sequencing 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: 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, 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, 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, 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, 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