Journal: Cell death & disease

Article Title: DEF6(differentially exprehomolog) exacerbates pathological cardiac hypertrophy via RAC1.

doi: 10.1038/s41419-023-05948-0

Figure Lengend Snippet: Fig. 1 The expression of DEF6 is increased in hypertrophic hearts and cardiomyocytes. A mRNA levels of DEF6 in the LV myocardium of mice subjected to sham or 4 weeks of TAC surgery (n = 5). B Immunoblot analyses (left) and results of quantification (right) of DEF6 protein expression in the LV myocardium of mice subjected to sham or 4 weeks of TAC surgery (n = 4). C mRNA levels of DEF6 in NRCMs administrated with PBS or 24 h of PE (50 μM) (n = 5). D Immunoblot analyses (left) and results of quantification (right) of DEF6 protein expression in NRCMs administrated with PBS or 24 h of PE (n = 4). *P < 0.05, ***P < 0.001 vs. sham or PBS. Data are displayed as mean ± SD. Statistical analysis were conducted by two-tailed Student’s t test (A, C) or Mann–Whitney U test (B, D).

Article Snippet: The cardiomyocytes were cultured in DMEM/F12 medium (Gibco, C11330) added with 10% fetal bovine serum (FBS), 5-bromodeoxyuridine (0.1 mM), and 1% penicillin/streptomycin for 24 h. The NRCMs were infected with adenoviruses at a multiplicity of infection (MOI) of 100 for 6 h. Subsequently, the medium was replaced with serum-free DMEM/F12, and 12 h later, the cardiomyocytes were stimulated with phosphatebuffered saline (PBS) or PE (50 μM) for 24 h. For rescue experiments, the cardiomyocytes were treated with Rac1 inhibitor NSC23766 (50 μM, 24 h) (S8031, Selleck) before adenovirus infection [31].

Techniques: Expressing, Western Blot, Two Tailed Test, MANN-WHITNEY

Journal: Cell death & disease

Article Title: DEF6(differentially exprehomolog) exacerbates pathological cardiac hypertrophy via RAC1.

doi: 10.1038/s41419-023-05948-0

Figure Lengend Snippet: Fig. 2 Ablation of DEF6 mitigates TAC-induced cardiac hypertrophy. A Strategy to construct KO mice and the sequencing results of WT and KO mice. B Protein levels of cardiac DEF6 in WT and KO mice (n = 5). C Comparisons of HW, HW/BW, LW/BW, and HW/TL in WT and KO mice subjected to sham or 4 weeks of TAC surgery (n = 10). D Left, gross hearts and H&E-stained LV sections of each groups. Scale bars, 0.3 cm and 50 μm, respectively. Right, Comparisons of cardiomyocyte cross-sectional area from groups (n = 6). E RT-PCR analyses of the hypertrophic markers in the indicated groups (n = 4). F–H Comparisons of the LVEDd, LVESd, LVPWd, FS, and EF values in WT and KO mice subjected to sham or 4 weeks of TAC surgery (n = 10). I Left, PSR-stained LV sections in WT and KO mice subjected to sham or 4 weeks of TAC surgery. Scale bars, 50 μm. Right, comparisons of LV collagen volume between groups (n = 6). J RT-PCR analysis of the fibrotic markers in each groups (n = 4). *P < 0.05, **P < 0.01, ***P < 0.001 vs. WT sham, #P < 0.05, ##P < 0.01, ###P < 0.001 vs. WT TAC. Data are displayed as mean ± SD. Statistical analysis were conducted by One-way ANOVA (C, D, F–I) or Kruskal–Wallis test (E, J).

Article Snippet: The cardiomyocytes were cultured in DMEM/F12 medium (Gibco, C11330) added with 10% fetal bovine serum (FBS), 5-bromodeoxyuridine (0.1 mM), and 1% penicillin/streptomycin for 24 h. The NRCMs were infected with adenoviruses at a multiplicity of infection (MOI) of 100 for 6 h. Subsequently, the medium was replaced with serum-free DMEM/F12, and 12 h later, the cardiomyocytes were stimulated with phosphatebuffered saline (PBS) or PE (50 μM) for 24 h. For rescue experiments, the cardiomyocytes were treated with Rac1 inhibitor NSC23766 (50 μM, 24 h) (S8031, Selleck) before adenovirus infection [31].

Techniques: Construct, Sequencing, Staining, Reverse Transcription Polymerase Chain Reaction

Journal: Cell death & disease

Article Title: DEF6(differentially exprehomolog) exacerbates pathological cardiac hypertrophy via RAC1.

doi: 10.1038/s41419-023-05948-0

Figure Lengend Snippet: Fig. 3 Overexpression of DEF6 aggravates TAC-induced cardiac hypertrophy. A mmunoblot analyses (left) and results of quantification (right) of DEF6 protein expression in the hearts of mice injected with AAV9-vector or AAV9-DEF6 (n = 4). B Comparisons of HW, HW/BW, LW/ BW, and HW/TL in AAV9-vector- and AAV9-DEF6-infected mice subjected to sham or 4 weeks of TAC surgery (n = 10). C Left, gross hearts and H&E-stained LV sections of each groups. Scale bars, 0.3 cm and 50 μm, respectively. Right, Comparisons of cardiomyocyte cross-sectional area between groups (n = 6). D RT-PCR analyses of the hypertrophic markers in each groups (n = 4). E–G Comparisons of LVEDd, LVESd, LVPWd, FS, and EF in AAV9-vector- and AAV9-DEF6-infected mice subjected to sham or 4 weeks of TAC surgery (n = 10). H Left, PSR-stained LV sections in AAV9-vector- and AAV9-DEF6-infected mice subjected to sham or 4 weeks of TAC surgery. Scale bars, 50 μm. Right, comparisons of LV collagen volume between groups (n = 6). I RT-PCR analyses of the fibrotic markers in each groups (n = 4). *P < 0.05, **P < 0.01, ***P < 0.001 vs. AAV9- vector or AAV9-vector sham, #P < 0.05, ##P < 0.01, ###P < 0.001 vs. AAV9-vector TAC. Data are displayed as mean ± SD. Statistical analysis were conducted by Mann–Whitney U test (A) or One-way ANOVA (B, C, E–H) or Kruskal–Wallis test (D, I).

Article Snippet: The cardiomyocytes were cultured in DMEM/F12 medium (Gibco, C11330) added with 10% fetal bovine serum (FBS), 5-bromodeoxyuridine (0.1 mM), and 1% penicillin/streptomycin for 24 h. The NRCMs were infected with adenoviruses at a multiplicity of infection (MOI) of 100 for 6 h. Subsequently, the medium was replaced with serum-free DMEM/F12, and 12 h later, the cardiomyocytes were stimulated with phosphatebuffered saline (PBS) or PE (50 μM) for 24 h. For rescue experiments, the cardiomyocytes were treated with Rac1 inhibitor NSC23766 (50 μM, 24 h) (S8031, Selleck) before adenovirus infection [31].

Techniques: Over Expression, Expressing, Injection, Plasmid Preparation, Infection, Staining, Reverse Transcription Polymerase Chain Reaction, MANN-WHITNEY

Journal: Cell death & disease

Article Title: DEF6(differentially exprehomolog) exacerbates pathological cardiac hypertrophy via RAC1.

doi: 10.1038/s41419-023-05948-0

Figure Lengend Snippet: Fig. 4 DEF6 exacerbates PE-induced cardiomyocyte hypertrophy. A Immunoblot analyses (left) and results of quantification (right) of DEF6 protein expression in cultured NRCMs infected with AdshRNA or AdshDEF6 (n = 3). B Left, immunofluorescence staining (α-actinin, red) in cultured NRCMs infected with AdshRNA or AdshDEF6 and administrated with PBS or 24 h of PE. Scale bar, 20 μm. Right, comparisons of the cardiomyocyte surface areas in cultured NRCVs of each groups (n ≥48 cells per group). C RT-PCR analysis of the hypertrophic markers in cultured NRCVs of each groups (n = 3). D Immunoblot analyses (left) and results of quantification (right) of DEF6 protein expression in cultured NRCVs infected with Advector or AdDEF6 (n = 3). E Left, immunofluorescence staining (α-actinin, red) in cultured NRCMs infected with Advector or AdDEF6 and administrated with PBS or 24 h of PE. Scale bar, 20 μm. Right, comparisons of the cardiomyocyte surface areas in cultured NRCVs of each groups (n ≥48 cells per group). F RT-PCR analyses of the hypertrophic markers in cultured NRCVs of each groups (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001 vs. AdshRNA or AdshRNA PBS or Advector or Advector PBS, #P < 0.05, ##P < 0.01, ###P < 0.001 vs. AdshRNA PE or Advector PE. Data are displayed as mean ± SD. Statistical analysis were conducted by two-tailed Mann–Whitney U test (A, D) or One-way ANOVA (B, E) or Kruskal–Wallis test (C, F).

Article Snippet: The cardiomyocytes were cultured in DMEM/F12 medium (Gibco, C11330) added with 10% fetal bovine serum (FBS), 5-bromodeoxyuridine (0.1 mM), and 1% penicillin/streptomycin for 24 h. The NRCMs were infected with adenoviruses at a multiplicity of infection (MOI) of 100 for 6 h. Subsequently, the medium was replaced with serum-free DMEM/F12, and 12 h later, the cardiomyocytes were stimulated with phosphatebuffered saline (PBS) or PE (50 μM) for 24 h. For rescue experiments, the cardiomyocytes were treated with Rac1 inhibitor NSC23766 (50 μM, 24 h) (S8031, Selleck) before adenovirus infection [31].

Techniques: Western Blot, Expressing, Cell Culture, Infection, Staining, Reverse Transcription Polymerase Chain Reaction, Two Tailed Test, MANN-WHITNEY

Journal: Cell death & disease

Article Title: DEF6(differentially exprehomolog) exacerbates pathological cardiac hypertrophy via RAC1.

doi: 10.1038/s41419-023-05948-0

Figure Lengend Snippet: Fig. 6 Prohypertrophic effect of DEF6 depends on Rac1-MEK-ERK signaling. A Co-IP of DEF6 was performed with anti-Flag and probed by Western blots with anti-HA (left); Co-IP of Rac1 was performed with anti-HA and probed by Western blots with anti-Flag (right). B In vitro GST pulldown assays for the interaction of purified Flag-DEF6 and GST-HA-Rac1 (left), as well as Flag-Rac1 and GST-HA-DEF6 (right). C The activity of Rac1 changes in the same direction as the DEF6 expression. D Immunoblot analyses of total and activated MEK1/2, ERK1/2 in cultured NRCMs infected with Advector or AdDEF6 and treated with PBS or NSC23766 (50 μM, 24 h) under 24 h of PE treatment (50 μM) (n = 3). E Immunofluorescence staining (α-actinin, red) (left) and comparison of cardiomyocyte surface areas (right) of NRCMs infected with Advector and AdDEF6 and treated with PBS or NSC23766 (50 μM, 24 h) under 24 h of PE treatment (50 μM). (n ≥48 cells per group). F RT-PCR analysis of the hypertrophic markers in cultured NRCVs of each groups (n = 3). G Immunoblot analyses of total and activated MEK1/2, ERK1/2 in cultured NRCMs infected with AdshRNA or AdshDEF6 and with Adcontrol or AdRac1(G12V) under PE 24 h of PE treatment (50 μM) (n = 3). H Immunofluorescence staining (α-actinin, red) (left) and comparison of cardiomyocyte surface areas (right) of NRCMs infected with the indicated adenovirus and administrated with 24 h of PE (50 μM) (n ≥48 cells per group). I RT-PCR analysis of the hypertrophic markers in cultured NRCVs of each groups (n = 3). •P < 0.05, P < 0.01,•P < 0.001 vs. Advector PBS PE or AdshRNA Adcontrol PE, *P < 0.05, **P < 0.01, ***P < 0.001 vs. Advector PBS PE or AdshRNA Adcontrol PE, ###P < 0.001 vs. AdDEF6 PBS PE or AdshDEF6 Adcontrol PE, and n.s. indicates no significance. Data are displayed as mean ± SD. Statistical analysis were conducted by Kruskal–Wallis test.

Article Snippet: The cardiomyocytes were cultured in DMEM/F12 medium (Gibco, C11330) added with 10% fetal bovine serum (FBS), 5-bromodeoxyuridine (0.1 mM), and 1% penicillin/streptomycin for 24 h. The NRCMs were infected with adenoviruses at a multiplicity of infection (MOI) of 100 for 6 h. Subsequently, the medium was replaced with serum-free DMEM/F12, and 12 h later, the cardiomyocytes were stimulated with phosphatebuffered saline (PBS) or PE (50 μM) for 24 h. For rescue experiments, the cardiomyocytes were treated with Rac1 inhibitor NSC23766 (50 μM, 24 h) (S8031, Selleck) before adenovirus infection [31].

Techniques: Co-Immunoprecipitation Assay, Western Blot, In Vitro, Activity Assay, Expressing, Cell Culture, Infection, Staining, Comparison, Reverse Transcription Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: NOD2/RICK-Dependent β-Defensin 2 Regulation Is Protective for Nontypeable Haemophilus influenzae -Induced Middle Ear Infection

doi: 10.1371/journal.pone.0090933

Figure Lengend Snippet: (A) Quantitative RT-PCR analysis shows that NOD2-specific siRNA inhibits NTHi lysate-induced human β-defensin 2 up-regulation more than NOD1-specific siRNA in the HMEEC cells. ELISA analysis shows that NTHi lysate-induced human β-defensin 2 production is suppressed by the NOD2-specific siRNA (B) but is enhanced by NOD2 overexpression (C) in the HMEEC cells. DEFB4: human β-defensin 2, NC: a control group silenced with a nonspecific negative control siRNA, KD: a group silenced with a gene-specific siRNA, pcDNA: a mock transfection, hNOD2: a construct expressing human NOD2. Results were expressed as fold induction, taking the value of the non-treated group as 1. The experiments were performed in triplicate and repeated twice. Values are given as the mean ± standard deviation (n = 3). *: p <0.05.

Article Snippet: Taqman primers and probes for human β-defensin 2 (DEFB4, NM_004942, Hs00175474_m1), TLR2 (NM_003264, Hs00152932_m1), NOD1 (NM_006092, Hs00196075_m1), NOD2 (NM_022162, Hs00223394_m1) and cyclophilin (NM_005729, 4326316E) were purchased from Life Technologies.

Techniques: Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Over Expression, Control, Negative Control, Transfection, Construct, Expressing, Standard Deviation

Journal: PLoS ONE

Article Title: NOD2/RICK-Dependent β-Defensin 2 Regulation Is Protective for Nontypeable Haemophilus influenzae -Induced Middle Ear Infection

doi: 10.1371/journal.pone.0090933

Figure Lengend Snippet: (A) Luciferase assays show that NTHi lysate-induced human β-defensin 2 up-regulation is enhanced by silencing of NLRC4 (a NOD2 inhibitor) but is inhibited by silencing of RICK that is downstream to NOD2 in the HMEEC cells. NC: a control group transfected with a nonspecific siRNA, KD: a group transfected with a gene-specific siRNA. (B) RT-PCR analysis showing siRNA-mediated inhibition of NLRC4 and RICK expression in the HMEEC cells. (C) Luciferase assays demonstrate that NTHi lysate-induced NF-κB activation is inhibited by the siRNAs specific to NOD2 and RICK in the HMEEC cells. pTAL-luc: a control vector containing the firefly luciferase gene with a TATA-like promoter region from the Herpes simplex virus thymidine kinase promoter, pNFκB-luc: a vector containing multiple copies of the NF-κB consensus sequence fused to pTAL-luc, Tx: treatment. Results were expressed as fold-induction, taking the value of the non-treated group as 1. The experiments were performed in triplicate and repeated twice. Values are given as the mean ± standard deviation (n = 3). *: p <0.05.

Article Snippet: Taqman primers and probes for human β-defensin 2 (DEFB4, NM_004942, Hs00175474_m1), TLR2 (NM_003264, Hs00152932_m1), NOD1 (NM_006092, Hs00196075_m1), NOD2 (NM_022162, Hs00223394_m1) and cyclophilin (NM_005729, 4326316E) were purchased from Life Technologies.

Techniques: Luciferase, Control, Transfection, Reverse Transcription Polymerase Chain Reaction, Inhibition, Expressing, Activation Assay, Plasmid Preparation, Virus, Sequencing, Standard Deviation

Journal: PLoS ONE

Article Title: NOD2/RICK-Dependent β-Defensin 2 Regulation Is Protective for Nontypeable Haemophilus influenzae -Induced Middle Ear Infection

doi: 10.1371/journal.pone.0090933

Figure Lengend Snippet: (A) Fluorescent microscopic images showing that α-hemolysin, a pore-forming toxin, enhances internalization of live NTHi (green) in the HMEEC cells. (B) An influx of calcein AM into the HMEEC cells was increased by α-hemolysin. (C) Quantitative RT-PCR analysis shows that α-hemolysin markedly enhances human β-defensin 2 up-regulation induced by a suboptimal dose (1 µg/ml) of NTHi lysate in the HMEEC cells. DEFB4: human β-defensin 2. (D) Note that silencing of NOD2 inhibits α-hemolysin-mediated enhancement of NTHi lysate-induced human β-defensin 2 up-regulation in the HMEEC cells. NC: a control group transfected with a nonspecific siRNA, KD: a group transfected with a gene-specific siRNA. Results were expressed as fold-induction, taking the value of the non-treated group as 1. The experiments were performed in triplicate and repeated twice. Values are given as the mean ± standard deviation (n = 3). *: p <0.05.

Article Snippet: Taqman primers and probes for human β-defensin 2 (DEFB4, NM_004942, Hs00175474_m1), TLR2 (NM_003264, Hs00152932_m1), NOD1 (NM_006092, Hs00196075_m1), NOD2 (NM_022162, Hs00223394_m1) and cyclophilin (NM_005729, 4326316E) were purchased from Life Technologies.

Techniques: Quantitative RT-PCR, Control, Transfection, Standard Deviation

Journal: PLoS ONE

Article Title: NOD2/RICK-Dependent β-Defensin 2 Regulation Is Protective for Nontypeable Haemophilus influenzae -Induced Middle Ear Infection

doi: 10.1371/journal.pone.0090933

Figure Lengend Snippet: (A) Quantitative RT-PCR analysis shows that NOD2-deficient middle ear epithelial cells less up-regulate mouse Defb2 in response to the NTHi molecules, compared to the wild type cells. Values are given as the mean ± standard deviation (n = 3). *: p <0.05. (B) Bacterial clearance analysis shows that the number of survived NTHi after intratympanic injection is higher in the NOD2-deficient mice compared to the wild type mice. It is noted that intratympanic NTHi replicates 10-fold in the NOD2/TLR2-deficient mice (NOD2 −/− TL2 −/− ). Values are given as the mean ± standard deviation (n = 20). (C) Histological analysis shows that middle ear inflammation in response to intratympanic injected NTHi is less severe in the NOD2-deficient mice (NOD2 −/− ) than that in the wild type mice (WT). Arrowheads: middle ear effusion with infiltration of inflammatory cells, M: middle ear cavity, Co: cochlea. Original magnification: x50.

Article Snippet: Taqman primers and probes for human β-defensin 2 (DEFB4, NM_004942, Hs00175474_m1), TLR2 (NM_003264, Hs00152932_m1), NOD1 (NM_006092, Hs00196075_m1), NOD2 (NM_022162, Hs00223394_m1) and cyclophilin (NM_005729, 4326316E) were purchased from Life Technologies.

Techniques: Quantitative RT-PCR, Standard Deviation, Injection

Journal: Calcified tissue international

Article Title: VWC2 Increases Bone Formation Through Inhibiting Activin Signaling

doi: 10.1007/s00223-018-0462-9

Figure Lengend Snippet: Effect of VWC2 on activin A-induced cell functions. a Effect of VWC2 on activin A-induced osteoblastic cell growth. MC3T3-E1 cells were plated in triplicate, and on the following day, cells were treated with PBS (control), activin A (250 ng/ml), VWC2 (500 ng/ml), or both VWC2 and activin A, and further cultured up to 14 days. Cell numbers were counted at each time point indicated and expressed as the mean ± SD. ***P value < 0.001, **P value < 0.01, *P value < 0.05. b Effect of VWC2 on osteoblast differentiation treated with activin A. MC3T3-E1 cells were treated with PBS (control), activin A (250 ng/ml), and VWC2 together with activin A, and further cultured for 7 days. Total RNA was extracted and the expression of osteoblastic markers was analyzed by real-time PCR. The normalized values are shown as mean + S.D. based on triplicate assays and statistically analyzed. **P value < 0.01, *P value < 0.05. Runx2; runtrelated transcription factor 2, Osx; Osterix, Col1a2; Collagen type 1 alpha 2 chain, Atf4; Activating transcription factor 4, Ocn; Osteocalcin

Article Snippet: Real-time PCR was performed using the following osteogenic markers: Runt-related transcription factor 2 (Runx2) / Core binding factor alpha 1 (Cbfa1) (Runx2 , Mm00501578_m1); Osterix ( Osx , Mm00504574_ m1); Collagen type 1 alpha 2 chain ( Col1a2 , Mm00483888_ m1); Activating transcription factor 4 ( Atf4 , Mm00515324_ m1); Bone γ-carboxyglutamate protein, related sequence 1 ( Osteocalcin / Ocn , Mm01741771_g1).

Techniques: Control, Cell Culture, Expressing, Real-time Polymerase Chain Reaction

Journal: Molecular Therapy. Nucleic Acids

Article Title: Effective Small Interfering RNA Therapy to Treat CLCN7 -dependent Autosomal Dominant Osteopetrosis Type 2

doi: 10.1038/mtna.2015.21

Figure Lengend Snippet: In vitro and in vivo tests of CLCN7 mutant -specific siRNAs . ( a ) Cartoon depicting the pEGFP-C1 vector used in the study. ( b ) HEK293 cells stably transfected with the pEGFP-C1 vector carrying the indicated mutations. Expression of the CLCN7 gene was quantified by real-time RT-PCR on RNA extracted from mutant transfectants, against cells transfected with the empty vector, which did not express CLCN7 mRNA (first bar from left). ( c–e ) HEK293 cells transfected with the indicated vectors, were treated with the CLCN7 mutant -specific siRNA listed in as the most effective per each mutation. Concentration-dependent regulation of CLCN7 assessed by real-time RT-PCR, normalized with GAPDH . ( f ) RT-PCR using primer pairs specific for the Clcn7 G213R mRNA showing transcript amplification only in heterozygous ( Clcn7 G213R/WT ) and homozygous ( Clcn7 G213R/G213R ) osteoclasts, while in wild-type osteoclasts ( Clcn7 WT/WT ) no transcript was amplified. ( g ) Direct DNA sequencing of the amplified transcript shown in f for the Clcn7 G213R/WT osteoclasts, demonstrating only the mutant sequence. ( h ) Osteoclasts generated from the bone marrow mononuclear cells of Clcn7 WT/WT and Clcn7 G213R/WT mice were treated with the indicated concentration of scrambled (SCR) or Clcn7 G213R -specific siRNA. Real-time RT-PCR was performed using the primer pairs specific for the mutant transcript validated in ( f ) and ( g ). ( i ) Osteoclasts were generated from the bone marrow mononuclear cells of Clcn7 WT/WT and Clcn7 G213R/WT mice onto bone slices and treated with the indicated concentration of SCR and Clcn7 G213R -specific siRNA. At the end of experiment, cells were removed by sonication and bone resorption evaluated by the pit assay. ( j ) Three-month-old Clcn7 WT/WT mice were injected once i.p. with 4 mg/kg of Clcn7 G213R -sticky siRNA jetPEI conjugate and sacrificed at the indicated time point. Sera were collected and evaluated for total RNA concentration by Nanodrop. ( k ) Ten-day-old Clcn7 G21R/WT mice were injected once i.p. with the indicated doses of SCR- or of Clcn7 G213R -sticky siRNA jetPEI conjugate. After 48 hours, mice were sacrificed, RNA was extracted from tibias, and evaluated by real-time RT-PCR using the primer pairs specific for the Clcn7 G213R mRNA validated in ( f ) and ( g ). In b–e, h–k data are the mean ± SD of three independent experiments or three animals/group. b–e,h,I,k : Student's t -test. j : one-way analysis of variance (ANOVA). For c–e , statistics was also performed by one way ANOVA (shown in Supplementary Table S3 ).

Article Snippet: The cationic polymer transfection reagent in vivo -jetPEI (cat# 201-50G) was from Polyplus-transfection (Illkirch, France).

Techniques: In Vitro, In Vivo, Mutagenesis, Plasmid Preparation, Stable Transfection, Transfection, Expressing, Quantitative RT-PCR, Concentration Assay, Reverse Transcription Polymerase Chain Reaction, Amplification, DNA Sequencing, Sequencing, Generated, Sonication, Injection

Journal: Molecular Therapy. Nucleic Acids

Article Title: Effective Small Interfering RNA Therapy to Treat CLCN7 -dependent Autosomal Dominant Osteopetrosis Type 2

doi: 10.1038/mtna.2015.21

Figure Lengend Snippet: In vivo treatment and safety study . Ten-day-old Clcn7 G213R/WT mice were injected i.p. with 4 mg/kg of Clcn7 G213R -sticky siRNA jetPEI conjugate, three times a week for 4 weeks. At the end of the experiments, mice were sacrificed and ( a ) the indicated organs were subjected to histopathological evaluation by hematoxylin/eosin staining (Bar = 100 µm for spleen and kidney, 20 µm for liver). ( b ) Sera were collected and analyzed by the Reflotron method for the indicated biomarkers of kidney and liver disease, and for the ADO2 biomarker CK. Normal values are between the two dotted lines. ( c ) RNA was extracted from the indicated organs and subjected to real time RT-PCR using primer pairs specific for the Clcn7 G213R mRNA, normalized for gapdh . ( d ) Ten day-old Clcn7 WT/WT and Clcn7 G213R/WT were treated with 4 mg/kg of scrambled- (SRC) or Clcn7 G213R -sticky siRNA jetPEI conjugate, three times a week for 2 and 4 weeks. At the end of the experiments, mice were sacrificed, then the serum biomarker of bone resorption, CTX, the serum osteoclast biomarker, TRAcP (5b isoform), and the CTX/TRAcP ratio were evaluated after 2 and 4 weeks of treatment. ( e ) µCT analysis of proximal tibias of mice treated with 4 mg/kg of Clcn7 G213R -sticky siRNA jetPEI conjugate, three times a week for 2 weeks, followed by measurements of trabecular ( f ) bone volume over total tissue volume (BV/TV), ( g ) trabecular number (Th.N), ( h ) thickness (Tb.Th), and ( i ) separation (Tb.Sp). Data are ( a,e ) representative or ( b–d, f–i ) the mean ± SD of four to seven mice per group (Student's t -test). For f–i statistics was also performed by one-way analysis of variance (shown in Supplementary Table S3 ).

Article Snippet: The cationic polymer transfection reagent in vivo -jetPEI (cat# 201-50G) was from Polyplus-transfection (Illkirch, France).

Techniques: In Vivo, Injection, Staining, Biomarker Discovery, Quantitative RT-PCR

Journal: Molecular Therapy. Nucleic Acids

Article Title: Effective Small Interfering RNA Therapy to Treat CLCN7 -dependent Autosomal Dominant Osteopetrosis Type 2

doi: 10.1038/mtna.2015.21

Figure Lengend Snippet: Rescue of the bone phenotype . Ten-day-old Clcn7 WT/WT and Clcn7 G213R/WT were treated with 4 mg/kg of scrambled- (SRC) or Clcn7 G213R -sticky siRNA jetPEI conjugate, three times a week for 4 weeks. At the end of the experiments, mice were sacrificed and their bone phenotype analyzed. ( a ) µCT analysis of proximal tibias. ( b ) Trabecular bone volume over total tissue volume (BV/TV). ( c ) Trabecular number (Tb.N). ( d ) Trabecular thickness (Tb.Th). ( e ) Trabecular separation (TB.Sp). ( f ) Serum concentration of ParaThyroid Hormone (PTH). ( g ) Histochemical TRAcP staining to evaluate osteoclasts (purple cells). Bar = 100 µm. ( h ) Osteoclast surface over bone surface (Oc.S/BS). ( i ) Osteoclast number over bone perimeter (Oc.N/B Pm). ( j ) Transcriptional expression, by real-time RT-PCR on RNA extracted from the whole femurs of osteoclast ( Tracp and Cathepsin K ( CatK )) and osteoblast ( Alkaline phosphatase ( ALP ) and Runt-related transcription factor 2 ( Runx 2 )) genes normalized with gapdh . ( k ) Eroded surface over bone surface (ES/BS). ( l ) Representative images of the secondary spongiosa (upper panels) and measurement of cartilage area/trabecular area (lower panel). Arrows: cartilage remnants. Bar = 50 µm. Results are ( a,g,i (upper panels)) representative or ( b–f,h–i (lower panel)) the mean ± SD of three to seven mice/group (Student's t -test). In d , P > 0.2. For b–f,h–l statistics was also performed by one-way analysis of variance (shown in Supplementary Table S3 ).

Article Snippet: The cationic polymer transfection reagent in vivo -jetPEI (cat# 201-50G) was from Polyplus-transfection (Illkirch, France).

Techniques: Concentration Assay, Staining, Expressing, Quantitative RT-PCR

Journal: Molecular Therapy. Nucleic Acids

Article Title: Effective Small Interfering RNA Therapy to Treat CLCN7 -dependent Autosomal Dominant Osteopetrosis Type 2

doi: 10.1038/mtna.2015.21

Figure Lengend Snippet: Cortical, growth plate, osteoblast, dynamic, and bone quality variables . Ten-day-old Clcn7 WT/WT and Clcn7 G213R/WT were treated with 4 mg/kg of scrambled- (SRC) or Clcn7 G213R -sticky siRNA jetPEI conjugate, three times a week for 4 weeks. At the end of the experiments, mice were sacrificed and their bone phenotype analyzed. ( a ) Cortical thickness (Cor.Th). ( b ) Growth plate width. ( c ) Osteoblast surface over bone surface (Ob.S/BS). ( d ) Histological images of osteoid (arrows). Bar = 5 µm. ( e ) Osteoid volume over bone volume (OV/BV). ( f ) Calcein labeling (green fluorescence) of mineral deposition (double arrowheads). Bar = 2 µm. ( g ) Mineral apposition rate (MAR). ( h ) Mineralized surface over bone surface (MS/BS). ( i ) Bone formation rate (BFR). ( j ) Total indentation distance (TDI). ( k ) First-cycle indentation distance (ID). ( l ) Touchdown distance (TDD). Results are ( d,f ) representative or ( a–c,e,g–l ) the mean ± SD of three to seven mice/group (Student's t -test). In a–c,e,g–i , P > 0.2. For a–c,e,g–l statistics was also performed by one-way analysis of variance (shown in Supplementary Table S3 ).

Article Snippet: The cationic polymer transfection reagent in vivo -jetPEI (cat# 201-50G) was from Polyplus-transfection (Illkirch, France).

Techniques: Labeling, Fluorescence

Journal: Molecular endocrinology (Baltimore, Md.)

Article Title: COUP-TFI (chicken ovalbumin upstream promoter-transcription factor I) regulates cell migration and axogenesis in differentiating P19 embryonal carcinoma cells.

doi: 10.1210/mend.14.12.0562

Figure Lengend Snippet: Fig. 1. Mutant C1413S COUP-TFI Dimerizes with wtCOUP-TFI and Inhibits Its DNA Binding Activity A, mutCOUP-TFI interacts with wtCOUP-TFI in a yeast two-hybrid system. Y190 yeast cells were transformed with the different vectors as shown. The resulting b-galactosidase activities were assayed and are shown as Miller units. Each bar represent the mean 6 SEM of four values obtained in two independent experiments. B, mutCOUP-TFI is retained on a GST-wtCOUP-TFI matrix. 4 ml of 35S-labeled in vitro translated mutCOUP-TFI were allowed to interact either with GST alone (lane 2) or a GST-DCOUP-TFI

Article Snippet: Briefly, 4 mg of WCEs were preincubated with 1 mg poly(dI-dC), and eventually with competitors, in 20 ml of binding buffer (20 mM HEPES, pH 7.9, 1 mM dithiothreitol, 50 mM KCl, 10% glycerol, 2.5 mM MgCl2) at room temperature for 15 min. For supershifting experiments, 1 ml of antibodies against COUP-TFI or COUP-TFII (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) were preincubated for 30 min with WCEs before addition of binding buffer.

Techniques: Mutagenesis, Binding Assay, Activity Assay, Transformation Assay, Labeling, In Vitro

Journal: Molecular endocrinology (Baltimore, Md.)

Article Title: COUP-TFI (chicken ovalbumin upstream promoter-transcription factor I) regulates cell migration and axogenesis in differentiating P19 embryonal carcinoma cells.

doi: 10.1210/mend.14.12.0562

Figure Lengend Snippet: Fig. 2. Stable Expression of COUP-TFI in Transfected P19 EC Cells A, RT-PCR analysis of transgene expression. Control cells (pcDNA), wild-type (wt), or mutant (mut) COUP-TF expressing cells were grown for 24 h as a monolayer in the presence or absence of cAMP as indicated before RNA extraction and RT-PCR analysis of COUP-TFI and the invariant PO gene expression. Results show the ethidium bromide staining of a 2% agarose gel. B and C, EMSAs of control (pcDNA) and wtCOUP-TFI cells treated as aggregates for 48 h with cAMP. An asterisk indicates the putative Ear2/DNA complex (see text), an arrow shows the position of the COUP-TFI/DNA complex, and an open circle shows the position of the supershifted complex in the presence of COUP-TFI antibodies (Ab). The oligonucleotides indicated in panel C were added as competitors at a 20-fold molar excess. D, DR-1 binding activity in P19 cell aggregates treated with 1026 M RA for 48 h. E, DR-1 binding activities in the different cell lines cultured as aggregates for 48 h and treated as indicated with all-trans-retinoic acid (RA) and/or cAMP. Symbols are the same as in Fig. 1B. Note that the mutant COUP-TFI inhibits endogenous COUP-TFs binding to the probe.

Article Snippet: Briefly, 4 mg of WCEs were preincubated with 1 mg poly(dI-dC), and eventually with competitors, in 20 ml of binding buffer (20 mM HEPES, pH 7.9, 1 mM dithiothreitol, 50 mM KCl, 10% glycerol, 2.5 mM MgCl2) at room temperature for 15 min. For supershifting experiments, 1 ml of antibodies against COUP-TFI or COUP-TFII (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) were preincubated for 30 min with WCEs before addition of binding buffer.

Techniques: Expressing, Transfection, Reverse Transcription Polymerase Chain Reaction, Control, Mutagenesis, RNA Extraction, Gene Expression, Staining, Agarose Gel Electrophoresis, Binding Assay, Activity Assay, Cell Culture

Journal: Molecular endocrinology (Baltimore, Md.)

Article Title: COUP-TFI (chicken ovalbumin upstream promoter-transcription factor I) regulates cell migration and axogenesis in differentiating P19 embryonal carcinoma cells.

doi: 10.1210/mend.14.12.0562

Figure Lengend Snippet: Fig. 4. RT-PCR Analysis of the Expression of Selected Ret- inoid-Responsive Genes RT-PCR analysis was run with RNA extracted from aggre- gates of pcDNA, wt, and mutant COUP-TFI cells treated as indicated for 48 h. Bmp-4, Bone morphogenetic protein-4; E-cad, E-cadherin.

Article Snippet: Briefly, 4 mg of WCEs were preincubated with 1 mg poly(dI-dC), and eventually with competitors, in 20 ml of binding buffer (20 mM HEPES, pH 7.9, 1 mM dithiothreitol, 50 mM KCl, 10% glycerol, 2.5 mM MgCl2) at room temperature for 15 min. For supershifting experiments, 1 ml of antibodies against COUP-TFI or COUP-TFII (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) were preincubated for 30 min with WCEs before addition of binding buffer.

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Mutagenesis

Journal: Molecular endocrinology (Baltimore, Md.)

Article Title: COUP-TFI (chicken ovalbumin upstream promoter-transcription factor I) regulates cell migration and axogenesis in differentiating P19 embryonal carcinoma cells.

doi: 10.1210/mend.14.12.0562

Figure Lengend Snippet: Fig. 5. P19 Cell Endogenous COUP-TFs Control Axogenesis Aggregated cells were plated after a 3-day RA treatment and further cultured for 5 days in the presence or not of 1 mM cAMP before anti-NF200 immunohistochemistry (magnification, 3400). Note the absence of neurites in mutCOUP-TFI cells treated with cAMP, despite the presence of neurofilament (NF200)-positive cell bodies (dark staining). Photographs show details of the different cultures and are representative of the content of the entire plates.

Article Snippet: Briefly, 4 mg of WCEs were preincubated with 1 mg poly(dI-dC), and eventually with competitors, in 20 ml of binding buffer (20 mM HEPES, pH 7.9, 1 mM dithiothreitol, 50 mM KCl, 10% glycerol, 2.5 mM MgCl2) at room temperature for 15 min. For supershifting experiments, 1 ml of antibodies against COUP-TFI or COUP-TFII (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) were preincubated for 30 min with WCEs before addition of binding buffer.

Techniques: Control, Cell Culture, Immunohistochemistry, Staining

Journal: Molecular endocrinology (Baltimore, Md.)

Article Title: COUP-TFI (chicken ovalbumin upstream promoter-transcription factor I) regulates cell migration and axogenesis in differentiating P19 embryonal carcinoma cells.

doi: 10.1210/mend.14.12.0562

Figure Lengend Snippet: Fig. 6. COUP-TFI Promotes Neuronal Migration A, Aggregates of cells treated for 3 days with 1 mM RA were plated onto tissue culture-grade plastic and further cultured for 24 h before the RGDS and SDGRG peptides were added at the indicated concentrations. After an additional day in culture, cells were fixed and stained for the presence of NF200 by immunohistochemistry (magnification, 3220). Neurons and neurites were intensely stained and either spread on the top of astrocytes [wtCOUP-TFI (no peptide)], or packed together [mutCOUP-TFI (no peptide), or wtCOUP-TFI RGDS 0.1 mg/ml]. B, The percentage of aggregates presenting either migrating neurons out of the core of the aggregates or a completely spread organization was determined and is shown as the percentage of aggregates with neuron outgrowth in function of the cell lines, in the absence of peptide (control) or in the presence of SDGRG and RGDS peptides at 0.1 mg/ml. Results are shown as the mean 6 SEM for 150 aggregates of each cell line scored in three independent experiments.

Article Snippet: Briefly, 4 mg of WCEs were preincubated with 1 mg poly(dI-dC), and eventually with competitors, in 20 ml of binding buffer (20 mM HEPES, pH 7.9, 1 mM dithiothreitol, 50 mM KCl, 10% glycerol, 2.5 mM MgCl2) at room temperature for 15 min. For supershifting experiments, 1 ml of antibodies against COUP-TFI or COUP-TFII (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) were preincubated for 30 min with WCEs before addition of binding buffer.

Techniques: Migration, Cell Culture, Staining, Immunohistochemistry, Control

Journal: Molecular endocrinology (Baltimore, Md.)

Article Title: COUP-TFI (chicken ovalbumin upstream promoter-transcription factor I) regulates cell migration and axogenesis in differentiating P19 embryonal carcinoma cells.

doi: 10.1210/mend.14.12.0562

Figure Lengend Snippet: Fig. 8. COUP-TFI Regulates the Synthesis of Vitronectin A, RT-PCR analysis was run with RNA extracted from aggregates (agg.) or monolayer cell cultures (mon.) of pcDNA, wt, and mutant COUP-TFI cells treated as indicated for 48 h. B, Alignment of the human and mouse proximal promoter regions of the vitronectin gene (black dots indicate identical nucleotides between mouse and human, and dashes represent gaps that were introduced to maximize sequence homology). The conserved nuclear receptor (NR) half-binding sites are boxed as well as putative binding sites for other transcription factors. C, The mouse vitronectin promoter is activated by COUP-TFI in transient transfection assays. P19 cells were cotransfected with a mouse vitronectin promoter fragment (2528/147) linked to a luciferase coding sequence and the indicated amounts of wt or mutCOUP-TFI expression vectors. Results are shown as the mean 6 SEM (n 5 3) of the relative luciferase activities (raw luciferase activities divided by b-galactosidase activities).

Article Snippet: Briefly, 4 mg of WCEs were preincubated with 1 mg poly(dI-dC), and eventually with competitors, in 20 ml of binding buffer (20 mM HEPES, pH 7.9, 1 mM dithiothreitol, 50 mM KCl, 10% glycerol, 2.5 mM MgCl2) at room temperature for 15 min. For supershifting experiments, 1 ml of antibodies against COUP-TFI or COUP-TFII (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) were preincubated for 30 min with WCEs before addition of binding buffer.

Techniques: Reverse Transcription Polymerase Chain Reaction, Mutagenesis, Sequencing, Binding Assay, Transfection, Luciferase, Expressing

Journal: PLoS ONE

Article Title: CD44v4 Is a Major E-Selectin Ligand that Mediates Breast Cancer Cell Transendothelial Migration

doi: 10.1371/journal.pone.0001826

Figure Lengend Snippet: A: differential migration of breast cancer cells across HUVEC monolayers pre-activated with TNF-α. B: migration of MDA-MB-231 across HUVEC monolayers in the presence of anti-E-selectin, anti-ICAM-1, and anti-VLA-4 antibodies (25 µg/ml each), respectively. HUVEC monolayers were pre-incubated with endothelial medium containing 25 ng/ml TNF-α for 6 h, and migration was terminated after overnight incubation at 37°C. All data are expressed as the mean±SD ( n = 3) of a typical experiment and similar results were obtained in at least three separate experiments.

Article Snippet: The monoclonal antibodies used in the experiments and their resources: mouse anti-human E-selectin mAb (MAB575), mouse anti-human CD44H (clone 2C5), mouse anti-human CD44v3 (clone 3G5), mouse anti-human CD44v6 (clone 2F10) and mouse anti-human CD44v4/5 (clone 3D2) (R&D Systems, Minneapolis, MN); Anti-CD44v4 mAb (Lab Vision, Fremont, CA); Anti-human LFA-1 and anti-human VLA-4 mAbs (BD Pharmingen, San Diego, CA); Anti-sLe x mAb (Clone KM93) (Chemicon, Temecula, CA); HRP-conjugated goat anti-mouse and anti-rabbit IgG (Jackson ImmunoResearch Lab, West Grove, PA); Alexa Fluor 488(495/519) and Alexa Fluor 568(578/603)-conjugated secondary antibodies (Molecular Probes, Eugene, OR).

Techniques: Migration, Incubation

Journal: PLoS ONE

Article Title: CD44v4 Is a Major E-Selectin Ligand that Mediates Breast Cancer Cell Transendothelial Migration

doi: 10.1371/journal.pone.0001826

Figure Lengend Snippet: Trypsin-elicited breast cancer cells were surface labeled with soluble exE-selectin/Fc or Fc-only as detailed in the . The bound Fc recombinants were detected by Alexa Fluor 488-conjugated goat anti-rabbit Fc and measured by FACS. In separated experiments, tumor cells were treated with neuraminidase to remove surface sLe x moieties before exE-selectin/Fc binding.

Article Snippet: The monoclonal antibodies used in the experiments and their resources: mouse anti-human E-selectin mAb (MAB575), mouse anti-human CD44H (clone 2C5), mouse anti-human CD44v3 (clone 3G5), mouse anti-human CD44v6 (clone 2F10) and mouse anti-human CD44v4/5 (clone 3D2) (R&D Systems, Minneapolis, MN); Anti-CD44v4 mAb (Lab Vision, Fremont, CA); Anti-human LFA-1 and anti-human VLA-4 mAbs (BD Pharmingen, San Diego, CA); Anti-sLe x mAb (Clone KM93) (Chemicon, Temecula, CA); HRP-conjugated goat anti-mouse and anti-rabbit IgG (Jackson ImmunoResearch Lab, West Grove, PA); Alexa Fluor 488(495/519) and Alexa Fluor 568(578/603)-conjugated secondary antibodies (Molecular Probes, Eugene, OR).

Techniques: Labeling, Binding Assay

Journal: PLoS ONE

Article Title: CD44v4 Is a Major E-Selectin Ligand that Mediates Breast Cancer Cell Transendothelial Migration

doi: 10.1371/journal.pone.0001826

Figure Lengend Snippet: A: After blocking with 5% normal goat serum in PBS, breast cancer cells cultured in a tissue culture dish were incubated with exE-selectin/Fc (10 µg/ml) in blocking solution for 1 h at 4°C. Following three washes, cells were fixed with 3.5% paraformaldehyde (5 min, 20°C) and the bound exE-selectin/Fc was detected by Alexa Fluor 488-conjugated anti-rabbit Fc. Cell surface labeling with Fc-only served as a background control. B: To determine the localization of E-selectin ligand on metastatic tumor cell surfaces during migration, MDA-MB-231 cells were double labeled with soluble exE-selectin/Fc for E-selectin ligand (left panel) and rhodamine-conjugated phalloidin for F-actin (right panel). Bar, 20 µm.

Article Snippet: The monoclonal antibodies used in the experiments and their resources: mouse anti-human E-selectin mAb (MAB575), mouse anti-human CD44H (clone 2C5), mouse anti-human CD44v3 (clone 3G5), mouse anti-human CD44v6 (clone 2F10) and mouse anti-human CD44v4/5 (clone 3D2) (R&D Systems, Minneapolis, MN); Anti-CD44v4 mAb (Lab Vision, Fremont, CA); Anti-human LFA-1 and anti-human VLA-4 mAbs (BD Pharmingen, San Diego, CA); Anti-sLe x mAb (Clone KM93) (Chemicon, Temecula, CA); HRP-conjugated goat anti-mouse and anti-rabbit IgG (Jackson ImmunoResearch Lab, West Grove, PA); Alexa Fluor 488(495/519) and Alexa Fluor 568(578/603)-conjugated secondary antibodies (Molecular Probes, Eugene, OR).

Techniques: Blocking Assay, Cell Culture, Incubation, Labeling, Control, Migration

Journal: PLoS ONE

Article Title: CD44v4 Is a Major E-Selectin Ligand that Mediates Breast Cancer Cell Transendothelial Migration

doi: 10.1371/journal.pone.0001826

Figure Lengend Snippet: A: Gel-code staining of proteins eluted from an exE-selectin/Fc affinity column. A 170 kD protein indicated by the arrow was selected as a potential ligand of E-selectin in tumor cells since 130 kD and 50 kD protein bands were confirmed as exE-selectin/Fc and IgG heavy chain, respectively. B: matching of the 10 amino acid N-terminal peptide sequence (red) of the 170 kD purified protein with that of human CD44. C: Western blot analysis of five breast cancer cell lines with anti-CD44H antibody.

Article Snippet: The monoclonal antibodies used in the experiments and their resources: mouse anti-human E-selectin mAb (MAB575), mouse anti-human CD44H (clone 2C5), mouse anti-human CD44v3 (clone 3G5), mouse anti-human CD44v6 (clone 2F10) and mouse anti-human CD44v4/5 (clone 3D2) (R&D Systems, Minneapolis, MN); Anti-CD44v4 mAb (Lab Vision, Fremont, CA); Anti-human LFA-1 and anti-human VLA-4 mAbs (BD Pharmingen, San Diego, CA); Anti-sLe x mAb (Clone KM93) (Chemicon, Temecula, CA); HRP-conjugated goat anti-mouse and anti-rabbit IgG (Jackson ImmunoResearch Lab, West Grove, PA); Alexa Fluor 488(495/519) and Alexa Fluor 568(578/603)-conjugated secondary antibodies (Molecular Probes, Eugene, OR).

Techniques: Staining, Affinity Column, Sequencing, Purification, Western Blot

Journal: PLoS ONE

Article Title: CD44v4 Is a Major E-Selectin Ligand that Mediates Breast Cancer Cell Transendothelial Migration

doi: 10.1371/journal.pone.0001826

Figure Lengend Snippet: A: RT-PCR amplification and DNA sequence of CD44 extracellular domain from various breast cancer cells and colonic HT29 cells. B: Western blot analysis of the 170 kD purified protein with antibodies specific to sLe x moieties and different CD44 variant isoforms, respectively. C: Specific binding of the 170 kD purified protein (CD44v4) to E-selectin through sLe x moieties. The data are represented as the mean±SD of three different experiments.

Article Snippet: The monoclonal antibodies used in the experiments and their resources: mouse anti-human E-selectin mAb (MAB575), mouse anti-human CD44H (clone 2C5), mouse anti-human CD44v3 (clone 3G5), mouse anti-human CD44v6 (clone 2F10) and mouse anti-human CD44v4/5 (clone 3D2) (R&D Systems, Minneapolis, MN); Anti-CD44v4 mAb (Lab Vision, Fremont, CA); Anti-human LFA-1 and anti-human VLA-4 mAbs (BD Pharmingen, San Diego, CA); Anti-sLe x mAb (Clone KM93) (Chemicon, Temecula, CA); HRP-conjugated goat anti-mouse and anti-rabbit IgG (Jackson ImmunoResearch Lab, West Grove, PA); Alexa Fluor 488(495/519) and Alexa Fluor 568(578/603)-conjugated secondary antibodies (Molecular Probes, Eugene, OR).

Techniques: Reverse Transcription Polymerase Chain Reaction, Amplification, Sequencing, Western Blot, Purification, Variant Assay, Binding Assay

Journal: PLoS ONE

Article Title: CD44v4 Is a Major E-Selectin Ligand that Mediates Breast Cancer Cell Transendothelial Migration

doi: 10.1371/journal.pone.0001826

Figure Lengend Snippet: A and B: siRNA transfection-induced CD44v4 downregulation in MDA-MB-231 cells as shown by Western blot (A) and immunofluorescence labeling (B), respectively. C and D: reduction of MDA-MB-231 cell adhesion to immobilized recombinant exE-selectin/Fc (C) and TNF-α pre-activated HUVEC monolayers (D) after CD44v4 downregulation via siRNA. E: reduction of MDA-MB-231 cell transendothelial migration by CD44v4 downregulation. The data are representative of three independent experiments.

Article Snippet: The monoclonal antibodies used in the experiments and their resources: mouse anti-human E-selectin mAb (MAB575), mouse anti-human CD44H (clone 2C5), mouse anti-human CD44v3 (clone 3G5), mouse anti-human CD44v6 (clone 2F10) and mouse anti-human CD44v4/5 (clone 3D2) (R&D Systems, Minneapolis, MN); Anti-CD44v4 mAb (Lab Vision, Fremont, CA); Anti-human LFA-1 and anti-human VLA-4 mAbs (BD Pharmingen, San Diego, CA); Anti-sLe x mAb (Clone KM93) (Chemicon, Temecula, CA); HRP-conjugated goat anti-mouse and anti-rabbit IgG (Jackson ImmunoResearch Lab, West Grove, PA); Alexa Fluor 488(495/519) and Alexa Fluor 568(578/603)-conjugated secondary antibodies (Molecular Probes, Eugene, OR).

Techniques: Transfection, Western Blot, Immunofluorescence, Labeling, Recombinant, Migration