Journal: PLoS ONE

Article Title: Low-Dosage Inhibition of Dll4 Signaling Promotes Wound Healing by Inducing Functional Neo-Angiogenesis

doi: 10.1371/journal.pone.0029863

Figure Lengend Snippet: A) Vascular density is increased in granulation tissue of mice treated with sDll4-Fc, being directly proportional to the dosage increase, when compared to controls throughout the experiment. B) Vascular smooth muscle cell coverage is decreased about 3-fold in relation to PBS-injected control mice throughout the experimental days in both tested groups. C,D,E) Representative anti-PECAM and anti-SMA immunofluorescence images of neo-vasculature in granulation tissue of wounds treated with (D) 0,05 mg/kg or (E) 2,5 mg/kg compared with control mice injected with PBS (C), in days 2, 4 and 6. sDll4-Fc therapy leads to decreased smooth muscle cell recruitment and increased vascular density that is directly proportional to the administered dosage. * In graphics represents p<0,05.

Article Snippet: Rat monoclonal anti-mouse PECAM (BD Pharmingen, San Jose, CA) and rabbit polyclonal anti-mouse α-SMA (Abcam, Cambridge, UK) were used as primary antibodies and species-specific conjugated with Alexa Fluor 488 and 555 (Invitrogen, Carlsbad, CA) were engaged as secondary antibodies.

Techniques: Injection, Immunofluorescence

Journal: PLoS ONE

Article Title: Low-Dosage Inhibition of Dll4 Signaling Promotes Wound Healing by Inducing Functional Neo-Angiogenesis

doi: 10.1371/journal.pone.0029863

Figure Lengend Snippet: Anti-PECAM (red) and anti-SMA (green) immunofluorescence of A) PBS-, B) 0,05 mg/kg sDll4-Fc- or C) 2,5 mg/kg sDll4-Fc-treated mice D,E,F) Representative anti-PECAM immunofluorescence images of neo-vasculature in unaffected skin adjacent to the wound site. G) Vascular density in unaffected skin is not statistically different between PBS-, 0,05 mg/kg- and 2,5 mg/kg-treated mice. The same happens for perivascular cell coverage H). I–J) Hematoxylin and eosin staining of 20 µm cryosections of PBS-, 0,05 mg/kg- and 2,5 mg/kg-treated mice wounds depicting the wound size and unaffected adjacent skin at day 6.

Article Snippet: Rat monoclonal anti-mouse PECAM (BD Pharmingen, San Jose, CA) and rabbit polyclonal anti-mouse α-SMA (Abcam, Cambridge, UK) were used as primary antibodies and species-specific conjugated with Alexa Fluor 488 and 555 (Invitrogen, Carlsbad, CA) were engaged as secondary antibodies.

Techniques: Immunofluorescence, Staining

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Inactivation of CDK2 is synthetically lethal to MYCN over-expressing cancer cells

doi: 10.1073/pnas.0901418106

Figure Lengend Snippet: CDK2 inhibition causes apoptosis in MYCN-amplified neuroblastoma cells. (A) Three MYCN amplified neuroblastoma cell lines, 1 MYCN single copy cell line (s.c.) and exponentially growing fibroblasts were transfected with 21-bp double strand siRNA against CDK2 or GFP as control. Samples were harvested at 48 h and immunoblotted for CDK2, PARP, and β-actin. The PARP antibody recognizes total and cleaved PARP (lower band). Cleavage of PARP indicates activation of the apoptotic pathway. Light microscopy pictures were taken just before harvesting the cells. (B) The neuroblastoma cell line IMR32 was transfected with a pcDNA6/TR vector and a vector containing CDK2 shRNA under a Tet operator-controlled CMV promoter generating the inducible cell line IMR32-pcDNA6-CDK2sh. Doxycycline was added at time point 0 to induce CDK2 shRNA expression and silence CDK2. Cells were harvested at various time points and immunoblotted for CDK2, threonine 821-phosphorylated pRb, PARP, cleaved caspase 3, and β-actin. Cleavage of PARP and caspase 3 indicates activation of the apoptotic pathway. (C) A firefly luciferase vector containing 6 E2F-binding sites was transfected together with a renilla luciferase vector in IMR32-pcDNA6-CDK2sh. After transfection, doxycycline was added. Control samples were not treated. Dual-luciferase assays were performed after 48 and 72 h to measure the relative E2F activity. (D) IMR32-pcDNA6-CDK2sh and the IMR32-pcDNA6 from which this cell line was derived were grown in the presence or absence of doxycycline. Cells were harvested at various time points and counted using a Coulter counter. (E) IMR32 cells were infected with the lentiviral vector encoding either CDK2 shRNA or the control shRNA and harvested at various time points after infection. Samples were immunoblotted for CDK2, PARP, cleaved caspase 3, and β-actin. (F) A CDK2 cDNA expression vector and an empty vector were transfected in IMR32. Clones were isolated using neomycin selection and 1 empty vector control clone and 2 clones expressing ectopic CDK2 were selected for further analysis. Cells were treated with increasing concentrations of lentiviral CDK2 shRNA. Nontreated (NT) samples were also included. Samples were immunoblotted for CDK2, PARP, and β-actin.

Article Snippet: The following antibodies were used as primary antibodies: CDK2 clone 55 mouse monoclonal (BD Bioscience), PARP clone 4C10–5 mouse monoclonal (BD PharMingen), pRb (pT821) (Biosource), P53 clone DO-7 mouse monoclonal (Labvision), Cleaved caspase 3 (Asp-175) rabbit polyclonal (Cell Signaling Technology), MYCN mouse monoclonal (PharMingen), histone H3 mouse monoclonal (Upstate), and actin C2 mouse monoclonal (Santa Cruz).

Techniques: Inhibition, Amplification, Transfection, Activation Assay, Light Microscopy, Plasmid Preparation, shRNA, Expressing, Luciferase, Binding Assay, Activity Assay, Derivative Assay, Infection, Clone Assay, Isolation, Selection

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Inactivation of CDK2 is synthetically lethal to MYCN over-expressing cancer cells

doi: 10.1073/pnas.0901418106

Figure Lengend Snippet: Apoptosis after CDK2 inhibition in neuroblastoma is dependent on MYCN over-expression. (A) SHEP-21N, a neuroblastoma cell line containing MYCN under a Tet repressor, was cultured with and without tetracycline for 3 days and then re-plated on 6-well plates at a density of 6 × 104 cells per well and treated with CDK2 siRNA or a control siRNA. Cells were harvested 48 h after siRNA treatment and immunoblotted for CDK2, MYCN, PARP, cleaved caspase 3, and β-actin. (B) Light microscopy pictures were taken just before harvesting the cells. Samples with MYCN on and CDK2 off settings showed a >94% reduction in cell density compared with all control samples. (C) IMR32, a neuroblastoma cell line with MYCN amplification, was seeded on 6-cm plates at a density of 2.5 × 105 cells per plate and treated with control siRNA, CDK2 siRNA, MYCN siRNA, or combination of MYCN and CDK2 siRNA. Cells were harvested after 48 h and immunoblotted for CDK2, MYCN, PARP, cleaved caspase 3, and β-actin. (D) Light microscopy pictures were taken just before harvesting the cells. Samples with MYCN on and CDK2 off settings sowed a >85% reduction in cell density compared with all control samples.

Article Snippet: The following antibodies were used as primary antibodies: CDK2 clone 55 mouse monoclonal (BD Bioscience), PARP clone 4C10–5 mouse monoclonal (BD PharMingen), pRb (pT821) (Biosource), P53 clone DO-7 mouse monoclonal (Labvision), Cleaved caspase 3 (Asp-175) rabbit polyclonal (Cell Signaling Technology), MYCN mouse monoclonal (PharMingen), histone H3 mouse monoclonal (Upstate), and actin C2 mouse monoclonal (Santa Cruz).

Techniques: Inhibition, Over Expression, Cell Culture, Light Microscopy, Amplification

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Inactivation of CDK2 is synthetically lethal to MYCN over-expressing cancer cells

doi: 10.1073/pnas.0901418106

Figure Lengend Snippet: Apoptosis after CDK2 silencing in MYCN-amplified cell is P53-mediated. (A) IMR32-pcDNA6-CDK2sh with doxycycline and IMR32-pcDNA6 were harvested for RNA isolation at various time points after the addition of doxycycline. Also, transient siRNA experiments were performed in IMR32 using GFP siRNA, CDK2 siRNA, MYCN siRNA, or CDK2 and MYCN siRNA. RNA was isolated at 24 and 48 h after transfection. Affymetrix microarray profiling was performed for both the inducible shRNA time-course experiments and the transient siRNA experiments. Affymetrix expression levels are shown for the 6 most strongly regulated genes. (See Materials and Methods for the selection procedure.) (B) IMR32-pcDNA6-CDK2sh was seeded on 6-cm plates at a density of 2.5 × 105 cells per plate and treated with doxycycline to induce CDK2 shRNA. Cells were harvested at various time points. Samples were immunoblotted for CDK2, P53, and β-actin. (C) IMR32 was seeded on 6-cm plates at a density of 2.5 × 105 cells per plate and treated with control siRNA, CDK2 siRNA, MYCN siRNA, or a combination of MYCN and CDK2 siRNA. Cells were harvested after 48 h and immunoblotted for CDK2, MYCN, P53, and β-actin. (D) IMR32 was seeded as described under Fig. 3C and treated with CDK2 siRNA or GFP siRNA and harvested 48 h after transfection. Total lysates and nuclear lysates were isolated. Total lysates were stained with CDK2 and P53 and the nuclear lysates with P53 and histone H3 for loading control. (E) IMR32 was seeded on 6-well plates at a density of 1 × 105 cells per well and infected with a lentiviral vector encoding CDK2 shRNA, P53 shRNA, or both shRNAs. Non-coding SHC002 control shRNA was added to experiments with CDK2 or P53 shRNA only and in the control samples to equal the amount of shRNA in each sample. Also a non-transfected control was included (o). Protein was harvested after 48 h and immunoblotted for CDK2, P53, PARP, and β-actin.

Article Snippet: The following antibodies were used as primary antibodies: CDK2 clone 55 mouse monoclonal (BD Bioscience), PARP clone 4C10–5 mouse monoclonal (BD PharMingen), pRb (pT821) (Biosource), P53 clone DO-7 mouse monoclonal (Labvision), Cleaved caspase 3 (Asp-175) rabbit polyclonal (Cell Signaling Technology), MYCN mouse monoclonal (PharMingen), histone H3 mouse monoclonal (Upstate), and actin C2 mouse monoclonal (Santa Cruz).

Techniques: Amplification, Isolation, Transfection, Microarray, shRNA, Expressing, Selection, Staining, Infection, Plasmid Preparation

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Inactivation of CDK2 is synthetically lethal to MYCN over-expressing cancer cells

doi: 10.1073/pnas.0901418106

Figure Lengend Snippet: The CDK2-inhibiting small molecule roscovitine induces P53-mediated apoptosis in cells over-expressing MYCN. (A) IMR32 was seeded in on 6-cm plates at a density of 5 × 105 cells per plate and treated with increasing concentrations of roscovitine. Cells were harvested 6 h after treatment. Samples were immunoblotted for threonine 821 phosphorylated pRb, P53, PARP, cleaved caspase 3, and β-actin. (B) SHEP-21-N, a neuroblastoma cell line containing MYCN under a Tet repressor, was cultured with or without tetracycline for 3 days and then re-plated on 6-cm plates at a density of 2.5 × 105 cells per plate. After 24 h, roscovitine was added to an end concentration of 7.5 μM. Twenty-four hours after start of treatment cells were harvested and immunoblotted for MYCN, PARP, and β-actin. (C) Light microscopy pictures were taken just before harvesting the cells. The sample with MYCN expression and roscovitine treatment showed a decrease of >91% in cell density compared with all control samples.

Article Snippet: The following antibodies were used as primary antibodies: CDK2 clone 55 mouse monoclonal (BD Bioscience), PARP clone 4C10–5 mouse monoclonal (BD PharMingen), pRb (pT821) (Biosource), P53 clone DO-7 mouse monoclonal (Labvision), Cleaved caspase 3 (Asp-175) rabbit polyclonal (Cell Signaling Technology), MYCN mouse monoclonal (PharMingen), histone H3 mouse monoclonal (Upstate), and actin C2 mouse monoclonal (Santa Cruz).

Techniques: Expressing, Cell Culture, Concentration Assay, Light Microscopy