Journal: Gastroenterology

Article Title: Cysteine-rich domains of muc3 intestinal mucin promote cell migration, inhibit apoptosis, and accelerate wound healing.

doi: 10.1053/j.gastro.2006.09.006

Figure Lengend Snippet: Figure 6. Effect of Muc3 and MUC3A expression on apoptosis. (A) Western blotting using anti-Flag antibody. Lanes: stably transfected LoVo clone LhM3c14 cytoplasmic fraction (LhM3c14 cyt) and membrane fraction (LHM3C14 MEM), mock-transfected LoVo cell clone membrane fraction (LMOCK MEM), nontransfected LoVo membrane fraction (MEM). (B) Percentage change in apoptosis with () or without () TNF- (100 ng/mL) treatment for 48 hours. Cell lines included parental LoVo, LhM3c14, Lmock, and nontransfected LoVo cells pretreated with m3EGF1,2 (10 g/mL) or GST (5 g/mL) for 1 hour before addition of TNF-. Baseline apoptosis 3.9%. *P .016 vs LoVo (), ^P .009 vs GST, P .002 vs Lmock; n 2–3 plates/treatment and 3 fields counted/plate. (C) Percentage change in apoptosis in nontransfected LoVo cells with () or without () sequential interferon- and anti-Fas antibody treatment for 72 hours. Cells were pretreated with m3EGF1,2 (10 g/mL), m3EGF1 (10 g/mL), m3EGF2 (10 g/mL), GST (10 g/mL), EGF (10 ng/mL), or m3EGF1 (5 g/mL) m3EGF2 (5 g/mL) before additionofanti-Fasantibody.Baselineapoptosis1.1%and.9%forleftandrightpanels,respectively(ANOVA,P.001,withinbothpanels).*P.005vsLoVo, LoVo m3EGF1, m3EGF2 and GST controls (left panel). *P .004 vs LoVo and LoVo m3EGF1 m3EGF2 (right panel); n 2 plates/treatment and 2 fields counted/plate. (D) Percentage change in apoptosis with () or without () sequential interferon- and anti-Fas antibody treatment for 48 hours. Cell lines included LhM3c14andLmock(ANOVA,P.001).Baselineapoptosis2.3%.*P.025vsLmock()andLhMc14()n3plates/treatmentand3fieldscounted/plate. (E) LoVo cells were treated with increasing concentrations of the EGF-receptor inhibitor, tyrphostin (AG1478). This resulted in a dose-dependent reversal of the anti-apoptosis effect of recombinant EGF, but did not affect the anti-apoptosis effect of m3EGF1,2 (ANOVA, P .001). Baseline apoptosis 2.0%. *P .03 vs m3EGF1,2 (same tyrphostin concentrations). (Note that maximal apoptosis observed varied from experiment to experiment because of the limited half-life of the activity of the anti-Fas in storage).

Article Snippet: Apoptosis was measured in histologic specimens using a erminal deoxynucleotidyl transferase–mediated deoxyuridine riphosphate biotin nick-end labeling (TUNEL) assay (TACS.XL n Situ Apoptosis Detection Kit; R&D Systems), according to he manufacturer’s directions.

Techniques: Expressing, Western Blot, Stable Transfection, Transfection, Membrane, Recombinant, Activity Assay

Journal: Gastroenterology

Article Title: Cysteine-rich domains of muc3 intestinal mucin promote cell migration, inhibit apoptosis, and accelerate wound healing.

doi: 10.1053/j.gastro.2006.09.006

Figure Lengend Snippet: Figure 9. Effect of recombinant Muc3 protein on acetic acid–in- duced apoptosis. (A) Mean number of apoptotic cells determined by TUNEL assay in the distal colon of mice 30 hours after acetic acid administration in mice treated twice with 100 g m3EGF1,2 or control peptide 100 g BSA in PBS (n 10 mice each treatment). (B) Repre- sentative distal colon 30 hours after acetic acid and control enema treatments. TUNEL-positive nuclei are stained darkly. (C) Representa- tive distal colon 30 hours after acetic acid and m3EGF1,2 enema treat- ments (magnification, 200).

Article Snippet: Apoptosis was measured in histologic specimens using a erminal deoxynucleotidyl transferase–mediated deoxyuridine riphosphate biotin nick-end labeling (TUNEL) assay (TACS.XL n Situ Apoptosis Detection Kit; R&D Systems), according to he manufacturer’s directions.

Techniques: Recombinant, TUNEL Assay, Control, Staining

Journal: Circulation Research

Article Title: Regulation of Vascular Smooth Muscle Cell Turnover by Endothelial Cell–Secreted MicroRNA-126

doi: 10.1161/circresaha.113.280883

Figure Lengend Snippet: Figure 1. Atheroprotective shear stress (SS) to endothelial cells (ECs) suppresses microRNA-126 (miR-126) in the cocultured smooth muscle cells (SMCs). A, Schematic diagrams of EC/SMC coculture and flow system. B, Human umbilical vein ECs and human umbilical artery SMCs were seeded on the lower and upper sides of a membrane, respectively (EC/SMC). Controls had SMCs but no ECs on the other side (Ø/SMC). The temporal expression levels of primary (pri–miR-126) and mature (miR-126) forms of miR-126 were determined by quantitative reverse transcription–polymerase chain reaction. *P<0.05 vs Ø/SMC 0-hour static. C, The levels of miR-126 in the 24-hour static or sheared media. *P<0.05 vs static control. D, The levels of miR-126 in SMCs incubated with medium 199 supplemented with 2% fetal bovine serum (control [CL] media) or conditioned medium from static or sheared ECs (24-hour shearing) (EC-CM). *P<0.05 vs CL media. E, miR-126 expression in EC monoculture or SMC coculture for 24 hours. F, miR-126 expression in ECs exposed to laminar SS (LSS) for 24 hours. G, The levels of pri–miR-126 and miR-126 in EC coculture with SMCs under 24–hour static or LSS conditions.

Article Snippet: Human umbilical vein ECs (HUVECs) were cultured in medium 199 (Gibco) supplemented with 10% fetal bovine serum (FBS) (Omega Scientific) and 10% Endothelial Growth Medium (Cell Applications).

Techniques: Shear, Membrane, Expressing, Reverse Transcription, Polymerase Chain Reaction, Control, Incubation

Journal: Circulation Research

Article Title: Regulation of Vascular Smooth Muscle Cell Turnover by Endothelial Cell–Secreted MicroRNA-126

doi: 10.1161/circresaha.113.280883

Figure Lengend Snippet: Figure 2. Endothelial cells (ECs) and shear stress (SS) regulate expressions of microRNA-126 (miR-126) targets in smooth muscle cells (SMCs). A, The mRNA levels of forkhead box O3 (FOXO3), B-cell lymphoma 2 (BCL2), and insulin receptor substrate 1 (IRS1) in Argonaute2 (Ago2) immunocomplexes in SMCs 48 hours after transfection with microRNA-126 mimics (PRE126) or anti–microRNA-126 inhibitor (AM126). *P<0.05 vs cells transfected with negative controls (negative control mimics [PREC] or negative control inhibitor [AMC]). B, The relative luciferase levels in SMCs cotransfected with PRE126 (left), AM126 (right), or the negative control and luciferase reporter plasmids: empty vector only, wild-type (WT) 3′-untranslated region (3′UTR) of FOXO3, BCL2, or IRS1 and mutants of 3′UTR of FOXO3, BCL2, or IRS1. C, The relative luciferase levels in SMCs transfected with the indicated luciferase reporter plasmids and then incubated with control media or EC-conditioned media (CM). *P<0.05 vs cells transfected with empty vector. #P<0.05 vs cells transfected with plasmids harboring the WT 3′UTRs. D, The protein levels of target genes in SMCs monocultured (Ø/SMC) or cocultured with ECs (EC/SMC) kept under static condition or exposed to laminar shear stress (LSS). Images are representative of triplicate experiments with similar results.

Article Snippet: Human umbilical vein ECs (HUVECs) were cultured in medium 199 (Gibco) supplemented with 10% fetal bovine serum (FBS) (Omega Scientific) and 10% Endothelial Growth Medium (Cell Applications).

Techniques: Shear, Transfection, Negative Control, Luciferase, Plasmid Preparation, Incubation, Control

Journal: Circulation Research

Article Title: Regulation of Vascular Smooth Muscle Cell Turnover by Endothelial Cell–Secreted MicroRNA-126

doi: 10.1161/circresaha.113.280883

Figure Lengend Snippet: Figure 3. Endothelial cells (ECs) transfer exogenous microRNA-126 (miR-126) to smooth muscle cells (SMCs). SMCs were incubated with conditioned media (CM) derived from ECs transfected with biotinylated synthetic miR-126 (B-miR-126) or no-miR mock control (Mock) for 3 hours. A, The levels of B-miR-126 in ECs or the EC-CM were determined by pull-down experiments with streptavidin- agarose beads followed by quantitative reverse transcription–polymerase chain reaction, (B) and B-miR-126 in ECs and SMCs was detected with QDot-605–streptavidin conjugates. *P<0.05 vs mock control. C and D, SMCs were cocultured with ECs transfected with B-miR-126 or mock control for 24 hours; the levels of B-miR-126 in SMCs were determined by Northern blot (C) or pull-down experiments with streptavidin-agarose beads (D). *P<0.05 vs mock control.

Article Snippet: Human umbilical vein ECs (HUVECs) were cultured in medium 199 (Gibco) supplemented with 10% fetal bovine serum (FBS) (Omega Scientific) and 10% Endothelial Growth Medium (Cell Applications).

Techniques: Incubation, Derivative Assay, Transfection, Control, Reverse Transcription, Polymerase Chain Reaction, Northern Blot

Journal: Circulation Research

Article Title: Regulation of Vascular Smooth Muscle Cell Turnover by Endothelial Cell–Secreted MicroRNA-126

doi: 10.1161/circresaha.113.280883

Figure Lengend Snippet: Figure 4. Functional microRNA-126 (miR-126) transmission from endothelial cells (ECs) to smooth muscle cells (SMCs) is independent of vesicles. A, The levels of miR-126 in SMCs incubated with control (CL) media or the EC-conditioned media (CM) treated with DNase I, RNase, or proteinase K (PK) for 3 hours. *P<0.05 vs CL media. B, The levels of miR-126 in ECs transfected with control small interfering (si)RNA; siCL) or siRNA against Drosha (siDROSHA), Dicer (siDICER1) or Argonaute2 (siEIF2C2) or in SMCs incubated with CL media or EC-CM derived from those transfected ECs. C, The levels of miR-126 in SMCs incubated EC-CM derived from ECs transfected with anti–miR-126 inhibitor anti–miR-126 inhibitor (AM126), miR-126 mimics (PRE126), or the negative controls. *P<0.05 vs siCL or CL media. #P<0.05 vs the negative control inhibitor (AMC) or negative control mimics (PREC) EC-CM. D, The levels of miR-126 in SMCs cocultured with lung ECs isolated from wild-type (WT) or miR-126 knockout mice (KO). *P<0.05 vs Ø/SMC (SMC monocultured). EC-CM was ultracentrifuged to fractionate the components in spin-down pellets (Pellet; which was reconstituted with M199), or the remaining supernatant (Super). The levels of miR-126 in (E) the fractionated EC-CM or in (F) SMCs incubated with total or fractionated EC-CM for 3 hours. G, The transcript levels or (H), protein levels of indicated genes in SMCs incubated with the fractioned EC-CM. Images are representative of triplicates with similar results. *P<0.05 vs CL media.

Article Snippet: Human umbilical vein ECs (HUVECs) were cultured in medium 199 (Gibco) supplemented with 10% fetal bovine serum (FBS) (Omega Scientific) and 10% Endothelial Growth Medium (Cell Applications).

Techniques: Functional Assay, Transmission Assay, Incubation, Control, Transfection, Derivative Assay, Negative Control, Isolation, Knock-Out

Journal: Circulation Research

Article Title: Regulation of Vascular Smooth Muscle Cell Turnover by Endothelial Cell–Secreted MicroRNA-126

doi: 10.1161/circresaha.113.280883

Figure Lengend Snippet: Figure 5. Argonaute2 (Ago2) associates with microRNA-126 (miR-126) and facilitates its transmission. A, The levels of miR-126 in endothelial cell–conditioned media (EC-CM) immunocomplexes of Ago2 or Ago1 or control immunoglobin G (IgG). *P<0.05 vs IgG control. B, Smooth muscle cells (SMCs) were cocultured with bovine aortic ECs transfected with a plasmid encoding the eGPF-Ago2. The enhanced green fluorescent protein-Ago2 proteins were visualized in live ECs or SMCs by microscopy. C, The protection of miR-126 with preincubation of bovine serum albumin (BSA) or recombinant human Ago2 protein (rAgo2) for 30 minutes after RNase A digestion for 15 minutes. *P<0.05 vs miR-126 only. D, The enrichment of miR-126 in SMCs treated with miR-126 preincubated with BSA or rAgo2 for 3 hours. *P<0.05 vs miRs only. E, The levels of Ago2 in ECs (left) or EC-CM (right) under static or laminar shear stress (LSS) conditions for 24 hours. F, The levels of miR-126 in Ago2 immunocomplex in EC-CM under static of LSS conditions. Images are representative of triplicates with similar results. Semiquantification results are shown in the lower panels. *P<0.05 vs static.

Article Snippet: Human umbilical vein ECs (HUVECs) were cultured in medium 199 (Gibco) supplemented with 10% fetal bovine serum (FBS) (Omega Scientific) and 10% Endothelial Growth Medium (Cell Applications).

Techniques: Transmission Assay, Control, Transfection, Plasmid Preparation, Microscopy, Recombinant, Shear

Journal: Circulation Research

Article Title: Regulation of Vascular Smooth Muscle Cell Turnover by Endothelial Cell–Secreted MicroRNA-126

doi: 10.1161/circresaha.113.280883

Figure Lengend Snippet: Figure 6. Shear stress (SS)–imposed endothelial cells (ECs) modulate smooth muscle cell (SMC) turnover through microRNA-126 (miR-126). SMCs were monocultured or cocultured with (1) ECs kept under static condition or exposed to laminar shear stress (LSS) for 24 hours; or (2) ECs transfected with negative control inhibitor (AMC) or anti–miR-126 inhibitor (AM126). A, Proliferating cell nuclear antigen (PCNA) in the SMCs was detected by immunostaining. B, Protein levels of PCNA, cyclinA, and p21 in SMCs were assayed by Western blot. C and D, SMC proliferation and apoptosis analyzed for propidium iodide–stained DNA content and Annexin V–stained membrane flip, respectively. *P<0.05 vs Ø/SMC (SMC monocultured). #P<0.05 vs Static EC-SMC or AMC EC-SMC.

Article Snippet: Human umbilical vein ECs (HUVECs) were cultured in medium 199 (Gibco) supplemented with 10% fetal bovine serum (FBS) (Omega Scientific) and 10% Endothelial Growth Medium (Cell Applications).

Techniques: Shear, Transfection, Negative Control, Immunostaining, Western Blot, Staining, Membrane

Journal: Circulation Research

Article Title: Regulation of Vascular Smooth Muscle Cell Turnover by Endothelial Cell–Secreted MicroRNA-126

doi: 10.1161/circresaha.113.280883

Figure Lengend Snippet: Figure 7. Depletion of microRNA-126 (miR-126) in mice inhibits smooth muscle cell (SMC) proliferation and neointimal formation in arteries after cessation of blood flow. A, The miR-126 expression levels in mouse carotid arteries with or without ligation. Vascular endothelial cells (ECs) were denuded before RNA isolation. *P<0.05 vs unligated control. B, In situ hybridization of miR-126 in ligated or unligated carotid arteries from miR-126 wild-type (WT) mice. Yellow arrows, elastic lamina (EL). C, Representative images of modified Verhoeff-Van Gieson–stained ligated or unligated carotid arteries from WT or miR-126 knockout (KO) mice. Arrows indicate the thickened neointima. D and E, Representative images of immunofluorescent-stained proliferating (proliferating cell nuclear antigen [PCNA]) SMC (smooth muscle-α actin) in ligated or unligated carotid arteries from WT and KO mice. F, Representative images of modified Verhoeff-Van Gieson–stained ligated carotid arteries from WT or KO mice treated with control (CL) media or EC-conditioned media (EC-CM); or with local delivery of CL oligo or miR-126 mimics (PRE126). Arrows indicate thickened neointima. G, In situ hybridization of miR-126 in ligated carotid arteries from KO mice with treatment of EC-CM, miR-126, or the respective controls.

Article Snippet: Human umbilical vein ECs (HUVECs) were cultured in medium 199 (Gibco) supplemented with 10% fetal bovine serum (FBS) (Omega Scientific) and 10% Endothelial Growth Medium (Cell Applications).

Techniques: Expressing, Ligation, Isolation, Control, In Situ Hybridization, Modification, Staining, Knock-Out

Journal: Journal of leukocyte biology

Article Title: Novel PKC signaling is required for LPS-induced soluble Flt-1 expression in macrophages.

doi: 10.1189/jlb.1007691

Figure Lengend Snippet: Fig. 2. GF109203X suppressed sFlt-1 ex- pression in LPS-activated RAW264.7 cells (A), which were pretreated with various con- centrations of the general PKC inhibitor GF109203X (0.05–5 M) for 1 h and sub- sequently stimulated with LPS for 24 h. The quantity of sFlt-1 in the conditioned media was measured using ELISA. Data are pre- sented as mean SEM (n3). (B) After pretreatment with GF109203X (5 M), RAW264.7 cells were stimulated with LPS for 8 h, and total RNA was harvested for determining sFlt-1 expression by real-time qRT-PCR assays. The data were normalized by GAPDH expression and presented as mean SEM (n3).

Article Snippet: The concentrations of sFlt-1 and free-form VEGF in each conditioned medium were analyzed using the mouse or human-specific ELISA kit (R&D Systems, Minneapolis, MN, USA).

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Quantitative RT-PCR

Journal: Journal of leukocyte biology

Article Title: Novel PKC signaling is required for LPS-induced soluble Flt-1 expression in macrophages.

doi: 10.1189/jlb.1007691

Figure Lengend Snippet: Fig. 3. GF109203X increased VEGF ex- pression in LPS-activated RAW264.7 cells (A), which were pretreated with various con- centrations of the general PKC inhibitor GF109203X (0.05–5 M) for 1 h followed by LPS stimulation for 24 h. The quantity of VEGF in the conditioned media was mea- sured using ELISA. Data are presented as mean SEM (n3). (B) After pretreatment with GF109203X (5 M), RAW264.7 cells were stimulated with LPS for 8 h, and total RNAs were harvested for determining VEGF expression by real-time qRT-PCR assays. The data were normalized by GAPDH expression and presented as mean SEM (n3).

Article Snippet: The concentrations of sFlt-1 and free-form VEGF in each conditioned medium were analyzed using the mouse or human-specific ELISA kit (R&D Systems, Minneapolis, MN, USA).

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Quantitative RT-PCR

Journal: Journal of leukocyte biology

Article Title: Novel PKC signaling is required for LPS-induced soluble Flt-1 expression in macrophages.

doi: 10.1189/jlb.1007691

Figure Lengend Snippet: Fig. 5. Effects of overexpressing dominant-negative mutant K376R of PKC on LPS-induced sFlt-1 and VEGF expressions in RAW264.7 macrophages. RAW264.7 cells containing the stably transfected pcDNA3.1 vector (3.1) or a dominant-negative mutant of PKC (K376R) were used. (A and C) These cells were treated with or without LPS (1 g/ml) for 15 h, and the total RNAs were collected for sFlt-1 and VEGF measurement by real-time qRT-PCR assays. The data were normalized by GAPDH expression and presented as mean SEM (n3). (B and D) These cells were treated with or without LPS (1 g/ml) for 24 h, and the quantity of sFlt-1 and VEGF in the conditioned media was measured by ELISA. Data are presented as mean SEM (n3).

Article Snippet: The concentrations of sFlt-1 and free-form VEGF in each conditioned medium were analyzed using the mouse or human-specific ELISA kit (R&D Systems, Minneapolis, MN, USA).

Techniques: Dominant Negative Mutation, Stable Transfection, Transfection, Plasmid Preparation, Quantitative RT-PCR, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Journal of leukocyte biology

Article Title: Novel PKC signaling is required for LPS-induced soluble Flt-1 expression in macrophages.

doi: 10.1189/jlb.1007691

Figure Lengend Snippet: Fig. 6. Effects of GF109203X on LPS- induced sFlt-1 and VEGF secretion in pri- mary human macrophages, which were pre- treated with 5 M GF109203X or DMSO as vehicle alone for 1 h and subsequently stim- ulated with LPS for 48 h. Human-specific ELISA kits were then used to detect sFlt-1 (A) and VEGF (B) in the collected condi- tioned media. Data are presented as mean SEM (n3).

Article Snippet: The concentrations of sFlt-1 and free-form VEGF in each conditioned medium were analyzed using the mouse or human-specific ELISA kit (R&D Systems, Minneapolis, MN, USA).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Oncogene

Article Title: Tamoxifen and TRAIL synergistically induce apoptosis in breast cancer cells.

doi: 10.1038/sj.onc.1210749

Figure Lengend Snippet: Figure 1 Effects of tamoxifen (TAM) and tumor necrosis factor- related apoptosis-inducing ligand (TRAIL) on apoptosis in breast cancer and normal epithelial cells. ER-a-positive (MCF-7, T47-D) and ER-a-negative (MDA-MB-231, BT-20) human breast cancer cells as well as normal breast epithelial cells (NBEC) were treated in serum-free medium with 2 mM TAM and/or 1 ng ml1 TRAIL for 24 h. (a) Hoechst staining of MCF-7 cells. Apoptotic nuclei are condensed or fragmented. (b) Percentage of apoptotic cells was determined after Hoechst staining as described (Chopin et al., 2004). Values represent mean7s.d. of three experiments. Syner- gism was calculated as described (Supplementary Data).

Article Snippet: Data represent mean7s.d. (n1⁄4 8). (b and c) Analysis of apoptosis after TUNEL staining (ApopTag Peroxidase in situ Apoptosis Detection Kit, R&D systems, Lille, France). (d) Analysis of cell proliferation after PCNA staining (Santa Cruz, Le Perray en Yvelines, France).

Techniques: Staining

Journal: Oncogene

Article Title: Tamoxifen and TRAIL synergistically induce apoptosis in breast cancer cells.

doi: 10.1038/sj.onc.1210749

Figure Lengend Snippet: Figure 2 Modulations of apoptosis regulating proteins by cotreatment with tamoxifen (TAM) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). MCF-7 and MDA-MB-231 breast cancer cells were treated in serum-free medium with 2 mM TAM and/or 1 ng ml1 TRAIL for 24 h. Western blot and confocal microscopy were then performed as described (Chopin et al., 2004). (a) Western blot analysis of whole cell lysates. (b) Confocal microscopy analysis of cytochrome c in MCF-7 cells. Typical punctuated mitochondrial staining was observed in control cells. A diffuse cytosolic signal was observed in cells treated with TRAIL and TAM alone, or in combination, suggesting the translocation of cytochrome c from mitochondria to cytosol. (c) Western blot analysis of mitochondrial and cytosolic cytochrome c in MCF-7 cells.

Article Snippet: Data represent mean7s.d. (n1⁄4 8). (b and c) Analysis of apoptosis after TUNEL staining (ApopTag Peroxidase in situ Apoptosis Detection Kit, R&D systems, Lille, France). (d) Analysis of cell proliferation after PCNA staining (Santa Cruz, Le Perray en Yvelines, France).

Techniques: Western Blot, Confocal Microscopy, Staining, Control, Translocation Assay

Journal: Oncogene

Article Title: Tamoxifen and TRAIL synergistically induce apoptosis in breast cancer cells.

doi: 10.1038/sj.onc.1210749

Figure Lengend Snippet: Figure 3 Effects of FADD-siRNA and Bid-siRNA on synergistic apoptosis induction. MCF-7 and MDA-MB-231 cells were subjected to one round of transfection with FADD-specific siRNA (a) or two sequential rounds of transfection with Bid-specific siRNA (b) as described (Grambihler et al., 2003; Broaddus et al., 2005). Twenty-four hours after transfection, cells were treated in serum-free medium with 2 mM tamoxifen (TAM) and 1 ng ml1 tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) alone or both for 24 h. Results were from one experiment representative of three independent experiments. *Po0.05 (Student’s t-test).

Article Snippet: Data represent mean7s.d. (n1⁄4 8). (b and c) Analysis of apoptosis after TUNEL staining (ApopTag Peroxidase in situ Apoptosis Detection Kit, R&D systems, Lille, France). (d) Analysis of cell proliferation after PCNA staining (Santa Cruz, Le Perray en Yvelines, France).

Techniques: Transfection

Journal: Oncogene

Article Title: Tamoxifen and TRAIL synergistically induce apoptosis in breast cancer cells.

doi: 10.1038/sj.onc.1210749

Figure Lengend Snippet: Figure 4 Effects of cotreatment with tamoxifen (TAM) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) on tumor growth in SCID mice. The ER-a-negative breast cancer cells MDA-MB-231 (2 106) were injected subcutaneously into the right flank of 6-week-old SCID mice (Charles River Laboratories. L’Arbresle, France). Tumor volumes were monitored every 3 days with calipers and calculated using the formula: 4/3p r12 r2, where r1 and r2 are the minor and the major dimensions, respectively. When tumor volume attained 0.6 cm3, the animals were grouped (n ¼ 8 for each group) and treated with an intraperitoneal injection of TRAIL (30 mg in 100 ml PBS) and/or TAM (250 mg in 100 ml corn oil) every other day for 15 days. (a) Tumor volumes at different times of treatment. Data represent mean7s.d. (n ¼ 8). (b and c) Analysis of apoptosis after TUNEL staining (ApopTag Peroxidase in situ Apoptosis Detection Kit, R&D systems, Lille, France). (d) Analysis of cell proliferation after PCNA staining (Santa Cruz, Le Perray en Yvelines, France). Significant differences (Student’s t-test) between treated and untreated mice are indicated with asterisks. *Po0.05; **Po0.01.

Article Snippet: Data represent mean7s.d. (n1⁄4 8). (b and c) Analysis of apoptosis after TUNEL staining (ApopTag Peroxidase in situ Apoptosis Detection Kit, R&D systems, Lille, France). (d) Analysis of cell proliferation after PCNA staining (Santa Cruz, Le Perray en Yvelines, France).

Techniques: Injection, TUNEL Assay, Staining, In Situ