Journal: Journal of Virology

Article Title: Host Response to the Attenuated Poxvirus Vector NYVAC: Upregulation of Apoptotic Genes and NF-κB-Responsive Genes in Infected HeLa Cells

doi: 10.1128/jvi.80.2.985-998.2006

Figure Lengend Snippet: FIG. 2. NYVAC-induced apoptosis in HeLa cells. (A) Protein validation of microarray data by immunofluorescence analysis of the effect of NYVAC infection on caspase-9 distribution in HeLa cells. Mock-, WR-, or NYVAC-infected cells were incubated at the indicated times with an anti-caspase-9 antibody (Calbiochem) that recognizes the active form of caspase-9, followed by the appropriate conjugated secondary antibody and the ToPro reagent. The samples were analyzed by confocal immunofluorescence microscopy. (B) Activation of caspase-9 after WR, MVA, or NYVAC infection. Total proteins (100 g) were separated by SDS-PAGE, transferred to nitrocellulose, and immunoblotted at various times (6 and 16 hpi) with an anti-caspase-9 antibody (Oncogene) that recognizes the procaspase and the cleaved active form of caspase-9. Caspase-9 presented a proform (46 kDa) that it is cleaved into a heterodimer of 35-kDa and 10-kDa chains. The molecular sizes (in kilodaltons) of the proteins are indicated on the right. Actin levels revealed that the same amount of protein was loaded into each lane. (C) Apoptosis assays after WR, MVA, or NYVAC infection determined the amount of cytoplasmic histone-associated DNA by ELISA. HeLa cells grown in 12-well plates were infected (5 PFU/cell) with the viruses indicated and harvested at 24 hpi for determinations with an ELISA kit of the absorbance at 405 nm. As a positive control for apoptosis, we used VV-PKR-infected HeLa cells. Duplicate samples were measured in two independent experiments.

Article Snippet: Protein lysates (100 g) were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) on 14% or 8% gels, transferred to nitrocellulose membranes, and incubated with the following antibodies: anti-caspase-9 (Oncogene), -PARP (Cell Signaling), -actin (Santa Cruz), -E3L (a gift from B. L. Jacobs), -A14L (43), -NF- B1, which recognizes p105 and p50 (Santa Cruz), -I B (Santa Cruz), -ATF-3 (Santa Cruz), -initiation factor 2 -P (IF-2 -P; Biosource), and -eIF-2 (Santa Cruz), followed by secondary antibodies (mouse and rabbit peroxidase conjugates).

Techniques: Biomarker Discovery, Microarray, Infection, Incubation, Microscopy, Activation Assay, SDS Page, Enzyme-linked Immunosorbent Assay, Positive Control

Journal: Journal of Virology

Article Title: Host Response to the Attenuated Poxvirus Vector NYVAC: Upregulation of Apoptotic Genes and NF-κB-Responsive Genes in Infected HeLa Cells

doi: 10.1128/jvi.80.2.985-998.2006

Figure Lengend Snippet: FIG. 3. Apoptosis induction kinetics during NYVAC infection. (A) Time course of PARP-1 cleavage during NYVAC infection. HeLa cells were infected with NYVAC, and at the indicated times, total proteins (100 g) were separated by SDS-PAGE, transferred to nitrocellulose, and immunoblotted with anti-PARP-1, which recognizes both the full-length and the cleaved protein. An 89-kDa cleavage product of PARP-1 was observed at 16 hpi. As a measure of infection, we used immunoblotting to visualize the viral proteins E3L and A14L. The molecular sizes (in kilodaltons) of the proteins are indicated on the right. Actin levels showed that the same amount of protein was loaded in each lane. (B) Apoptotic phenotype of NYVAC-infected HeLa cells. Monolayer cultures of HeLa cells were left uninfected or infected (5 PFU/cell) with the different VV strains for 16 h, and the apoptotic phenotype was visualized by phase-contrast microscopy. NYVAC-infected cells showed characteristic apoptosis, defined by a rounded, floating, phase-bright, wrinkled shape.

Article Snippet: Protein lysates (100 g) were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) on 14% or 8% gels, transferred to nitrocellulose membranes, and incubated with the following antibodies: anti-caspase-9 (Oncogene), -PARP (Cell Signaling), -actin (Santa Cruz), -E3L (a gift from B. L. Jacobs), -A14L (43), -NF- B1, which recognizes p105 and p50 (Santa Cruz), -I B (Santa Cruz), -ATF-3 (Santa Cruz), -initiation factor 2 -P (IF-2 -P; Biosource), and -eIF-2 (Santa Cruz), followed by secondary antibodies (mouse and rabbit peroxidase conjugates).

Techniques: Infection, SDS Page, Western Blot, Microscopy

Journal: Journal of Virology

Article Title: Host Response to the Attenuated Poxvirus Vector NYVAC: Upregulation of Apoptotic Genes and NF-κB-Responsive Genes in Infected HeLa Cells

doi: 10.1128/jvi.80.2.985-998.2006

Figure Lengend Snippet: FIG. 4. NYVAC-induced NF-B signaling. (A) Validation of microarray data by NF-B protein levels and comparison between WR, MVA, and NYVAC infections. Total proteins (100 g) were separated by SDS-PAGE, transferred to nitrocellulose, and immunoblotted with anti-NF- B1 antibody at various times (6 and 16 hpi). Actin levels indicated that the same amount of protein was loaded into each lane. The molecular sizes (in kilodaltons) of the proteins are indicated on the right. (B) Autoradiogram of NF-B activity, determined by EMSA performed with nuclear extracts of mock-, MVA-, and NYVAC-infected HeLa cells at the indicated times. WR, nuclear extract from WR-infected HeLa cells at 16 hpi. The arrow indicates the 32P-labeled NF-B/DNA complex and the nonspecific signal, and the asterisk indicates the supershift of NF-B after incubation of the nuclear extracts with the 32P-labeled probe and anti-p50 antibody. (C) I degradation kinetics of WR-, MVA-, and NYVAC-infected HeLa cells. Cytoplasmic extracts were prepared, as previously described (45), from uninfected and poxvirus-infected (5 PFU/cell) HeLa cells. I levels were detected by immunoblotting at 4 hpi. The molecular sizes (in kilodaltons) of the proteins are indicated on the right. Actin levels indicated that the same amount of protein was loaded into each lane of the gel. (D) Levels of IL-6 secreted from WR-, MVA-, and NYVAC-infected HeLa cells (5 PFU/cell), as determined by ELISA. Protein levels of IL-6 in supernatants of infected cells were measured at 2, 6, and 16 hpi. Duplicate samples were measured in two independent experiments.

Article Snippet: Protein lysates (100 g) were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) on 14% or 8% gels, transferred to nitrocellulose membranes, and incubated with the following antibodies: anti-caspase-9 (Oncogene), -PARP (Cell Signaling), -actin (Santa Cruz), -E3L (a gift from B. L. Jacobs), -A14L (43), -NF- B1, which recognizes p105 and p50 (Santa Cruz), -I B (Santa Cruz), -ATF-3 (Santa Cruz), -initiation factor 2 -P (IF-2 -P; Biosource), and -eIF-2 (Santa Cruz), followed by secondary antibodies (mouse and rabbit peroxidase conjugates).

Techniques: Biomarker Discovery, Microarray, Comparison, SDS Page, Activity Assay, Infection, Labeling, Incubation, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Journal of Virology

Article Title: Host Response to the Attenuated Poxvirus Vector NYVAC: Upregulation of Apoptotic Genes and NF-κB-Responsive Genes in Infected HeLa Cells

doi: 10.1128/jvi.80.2.985-998.2006

Figure Lengend Snippet: FIG. 5. ATF-3 protein expression is induced by NYVAC infection. (A) Levels of ATF-3, phosphorylated eIF2 at Ser-51, and total eIF2 were measured by immunoblot analysis of extracts of HeLa cells after WR, MVA, and NYVAC infection. Total proteins (100 g) were separated by SDS-PAGE, transferred to nitrocellulose, and immuno- blotted with anti-ATF-3, anti-phosphorylated-eIF2, or anti-eIF2 an- tibody at various times (6 and 16 hpi). The molecular sizes (in kilo- daltons) of the proteins are indicated on the right. The antibody against ATF-3 recognizes two or three bands, depending on the run- ning conditions, perhaps as a result of posttranslational modifications of the transcription factor, which can be cell dependent. (B) Immuno- fluorescence analysis of the effect of NYVAC infection on ATF-3 distribution in HeLa cells. Mock-, WR-, and NYVAC-infected cells were labeled with anti-ATF-3 (Santacruz) at the indicated times, fol- lowed by the appropriate conjugated secondary antibody and ToPro reagent. The samples were analyzed by confocal immunofluorescence microscopy.

Article Snippet: Protein lysates (100 g) were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) on 14% or 8% gels, transferred to nitrocellulose membranes, and incubated with the following antibodies: anti-caspase-9 (Oncogene), -PARP (Cell Signaling), -actin (Santa Cruz), -E3L (a gift from B. L. Jacobs), -A14L (43), -NF- B1, which recognizes p105 and p50 (Santa Cruz), -I B (Santa Cruz), -ATF-3 (Santa Cruz), -initiation factor 2 -P (IF-2 -P; Biosource), and -eIF-2 (Santa Cruz), followed by secondary antibodies (mouse and rabbit peroxidase conjugates).

Techniques: Expressing, Infection, Western Blot, SDS Page, Labeling, Microscopy

Journal: The FASEB Journal

Article Title: Heparin-binding epidermal growth factor-like growth factor promotes neuroblastoma differentiation

doi: 10.1096/fj.201600828R

Figure Lengend Snippet: HBEGF promotes neuroblast differentiation in NB cells. A) Western blot for differentiation markers after 72 h of HBEGF treatment in BE2 and SK-N-AS. Densitometry for NF160 normalized to β-actin is shown as the percentage of control. B) Quantification of NF160 densitometry normalized to β-actin from 3 independent Western blots (5Y, BE2) or 8 independent Western blots (SK-N-AS) after 72-h treatment with 1 ng/ml HBEGF and presented as means ± sem. P < 0.001 (1-way ANOVA). *P < 0.05, **P < 0.01, ****P < 0.0001, 1-sample Student’s t test. C) Representative phase-contrast images of BE2 cells after 72 h of treatment with HBEGF. Arrows identify long neurites. Original magnification, ×10. Scale bar, 100 μM. D) Quantification of neurite length using NeuronJ after 72 h of treatment with HBEGF from 3 independent experiments. P < 0.01 (1-way ANOVA). *P < 0.05, 1-sample Student’s t test. E) Western blot for differentiation markers after 72 h of HBEGF (0.5 or 1 ng/ml), FGF2 (1 or 10 ng/ml), or ATRA (1 or 10 μM). Densitometry for NF160 normalized to β-actin is shown as the percentage of control. F) Western blot for differentiation markers after 72 h HBEGF (0, 0.25, 0.5, 0.75, 1, 2 ng/ml) and a neutralizing HBEGF antibody (nAb; 0.0075, 0.015, 0.03, 0.05, 0.1, or 0.5 μg/ml). Densitometry for NF160 normalized to β-actin is shown as the percentage of control. G) Western blot for β3-tubulin and HBEGF in SHEP stably expressing an NTC shRNA or shRNA to HBEGF (shHBEGF #1, #2). Densitometry for β3-tubulin normalized to β-actin is shown as the percentage of control. H) Linear regression analyses using the microarray meta-dataset (left) or the GSE49710 dataset (right).

Article Snippet: The following antibodies for differentiation and signaling markers were purchased from Cell Signaling: neurofilament 160 kD (NF160) (2838), β3-tubulin (5568), neuron-specific enolase (9536), growth-associated protein 43 (5307 and 8945), phospho-ERK1/2 (p-ERK1/2; 9101), ERK1/2 (4695), p21 (2947), phospho-AKT (p-AKT; 4058), AKT (4691), phospho-STAT3 (p-STAT3; 9145), STAT3 (9139), and EGFR (4267).

Techniques: Western Blot, Control, Stable Transfection, Expressing, shRNA, Microarray

Journal: The FASEB Journal

Article Title: Heparin-binding epidermal growth factor-like growth factor promotes neuroblastoma differentiation

doi: 10.1096/fj.201600828R

Figure Lengend Snippet: Schwannian stroma-derived HBEGF promotes neuroblast differentiation. A) Immunofluorescence in NB specimens using HBEGF (green) and S100 schwannian stroma (red) antibodies. DAPI nuclear stain in blue. Original magnification, ×40. Scale bar, 50 μM. B) Western blot for differentiation markers in 5Y after 72 h of coculture or treatment with conditioned medium from SHEP stably expressing an NTC shRNA construct or shRNA to HBEGF (shHBEGF #1, #2). Densitometry for NF160 normalized to β-actin is shown as the percentage of control.

Article Snippet: The following antibodies for differentiation and signaling markers were purchased from Cell Signaling: neurofilament 160 kD (NF160) (2838), β3-tubulin (5568), neuron-specific enolase (9536), growth-associated protein 43 (5307 and 8945), phospho-ERK1/2 (p-ERK1/2; 9101), ERK1/2 (4695), p21 (2947), phospho-AKT (p-AKT; 4058), AKT (4691), phospho-STAT3 (p-STAT3; 9145), STAT3 (9139), and EGFR (4267).

Techniques: Derivative Assay, Immunofluorescence, Staining, Western Blot, Stable Transfection, Expressing, shRNA, Construct, Control

Journal: The FASEB Journal

Article Title: Heparin-binding epidermal growth factor-like growth factor promotes neuroblastoma differentiation

doi: 10.1096/fj.201600828R

Figure Lengend Snippet: HSPGs and EGFR interact to promote HBEGF-mediated neuroblast differentiation. A) Western blots for differentiation markers after 72-h treatment with 10 ng/ml soluble (s)TβRIII or sCD44 or 100 ng/ml sGPC1, sGPC3, or sSDC3 in the absence or presence of 0.5 ng/ml HBEGF in 5Y cells. B) Densitometry for NF160 normalized to β-actin is shown as the percentage of control, a dosecourse of HBEGF in SK-N-AS after 96-h TβRIII knockdown. C) Densitometry for NF160 normalized to β-actin is shown as the percentage of control, 0.5 μg/ml heparin, ODSH, 2-O desulfated heparin (2DES), 6-O desulfated heparin (6DES), or N desulfated heparin (NDES) in the absence or presence of 0.5 ng/ml HBEGF in SK-N-AS. D) Densitometry for NF160 normalized to β-actin is shown as the percentage of control, EGFR inhibitors for 72 h followed by 48 h of 1 ng/ml HBEGF treatment. Densitometry for NF160 normalized to β-actin is shown as the percentage of control. E) In situ proximity ligation assay in SK-N-AS after 5 min of treatment with 1 ng/ml HBEGF or 1 ng/ml EGF. Original magnification, ×40. Scale bars, 50 μM. Normalized TβRIII/EGFR complexes per cell (75–100 cells/condition) from 6 independent experiments. P < 0.001 (1-way ANOVA). *P < 0.05, ****P < 0.0001, 1-sample Student’s t test. F) Analysis of event-free survival in the GSE49710 dataset stratified by the top and bottom 50% for HBEGF, then TGFBR3, then EGFR expression (left) and analysis of event-free survival stratified by the top and bottom 12.5% for HBEGF, then TGFBR3, then EGFR expression (middle) compared with stratification by MYCN amplification status (right). NA, nonamplified.

Article Snippet: The following antibodies for differentiation and signaling markers were purchased from Cell Signaling: neurofilament 160 kD (NF160) (2838), β3-tubulin (5568), neuron-specific enolase (9536), growth-associated protein 43 (5307 and 8945), phospho-ERK1/2 (p-ERK1/2; 9101), ERK1/2 (4695), p21 (2947), phospho-AKT (p-AKT; 4058), AKT (4691), phospho-STAT3 (p-STAT3; 9145), STAT3 (9139), and EGFR (4267).

Techniques: Western Blot, Control, Knockdown, In Situ, Proximity Ligation Assay, Expressing, Amplification

Journal: The FASEB Journal

Article Title: Heparin-binding epidermal growth factor-like growth factor promotes neuroblastoma differentiation

doi: 10.1096/fj.201600828R

Figure Lengend Snippet: HBEGF induces neuroblast differentiation via ERK and STAT3 signaling and up-regulation of ID1. A) Western blot in SK-N-AS for phosphorylated and total STAT3 or ERK1/2 after 72 h treatment with 1 ng/ml HBEGF. Densitometry for phosphorylated STAT3 or phosphorylated ERK1/2 normalized to β-actin is shown as the percentage of control. B) Western blot for ID1 in BE2 and SK-N-AS after 72 h treatment with a dosecourse of HBEGF. Densitometry for ID1 normalized to β-actin is shown as the percentage of control. C) Western blot for ID1 in SHEP stably expressing an NTC shRNA or shRNA to HBEGF (shHBEGF #1, #2). Densitometry for ID1 normalized to β-actin is shown as the percentage of control. D) Western blot in SK-N-AS after 96 h ID1 knockdown and 72 h HBEGF treatment. Densitometry for NF160 normalized to β-actin is shown as the percentage of control. E) Linear regression analyses using the microarray meta-dataset (left) or the GSE49710 dataset (right). F) Western blot for differentiation markers and ID1 after 24 h cotreatment with 1 ng/ml HBEGF and the indicated doses of U0126 or CI-1040. Densitometry for NF160 normalized to β-actin is shown as the percentage of control. G) Western blot for differentiation markers after 72 h expression of an empty-vector control (EV) or a dominant negative STAT3 (DN STAT3) construct and 48 h treatment with 1 ng/ml HBEGF or 48 h treatment with ruxolitinib and 24 h treatment with 1 ng/ml HBEGF. Densitometry for NF160 normalized to β-actin is shown as the percentage of control.

Article Snippet: The following antibodies for differentiation and signaling markers were purchased from Cell Signaling: neurofilament 160 kD (NF160) (2838), β3-tubulin (5568), neuron-specific enolase (9536), growth-associated protein 43 (5307 and 8945), phospho-ERK1/2 (p-ERK1/2; 9101), ERK1/2 (4695), p21 (2947), phospho-AKT (p-AKT; 4058), AKT (4691), phospho-STAT3 (p-STAT3; 9145), STAT3 (9139), and EGFR (4267).

Techniques: Western Blot, Control, Stable Transfection, Expressing, shRNA, Knockdown, Microarray, Plasmid Preparation, Dominant Negative Mutation, Construct

Journal: International Journal of Molecular Sciences

Article Title: Cell Shape and Matrix Stiffness Impact Schwann Cell Plasticity via YAP/TAZ and Rho GTPases

doi: 10.3390/ijms22094821

Figure Lengend Snippet: RhoA and YAP/TAZ inhibition led to increased expression of regenerative markers c-Jun, p75NTR, and Sox-2 through the activation of Rac1/MKK7/JNK signaling. ( A – C ) Active Rac1 inhibition via siRac1 significantly lowers c-Jun and p75NTR expression with RhoA and YAP/TAZ inhibition, showing no change in expression when Rac1 activity is blocked by siRac1 transfection. Four cell lysates were created for Western blot quantification. ( D – F ) Immunofluorescent stained Schwann cells treated with a combination of Y-27632, Verteporfin, or siRac1 showing expression of p75NTR and Sox-2. Three unique trials with a minimum of 42 cells per trial were quantified. Scale bar = 50 μm. ( G ) Rac1/MKK7/JNK expression when treated with Y-27632 or Verteporfin. Data are presented as the mean ± SEM. ** p < 0.005, *** p < 0.0005, p -values found in .

Article Snippet: Y-27632 (Tocris, 1254) (20 μM) was used to inhibit Rho-associated, coiled-coil-containing protein kinase (ROCK) activity, and Verteporfin (Tocris, 5305) (1 μM) was used to inhibit active (nuclear) YAP/TAZ.

Techniques: Inhibition, Expressing, Activation Assay, Activity Assay, Transfection, Western Blot, Staining

Journal: International Journal of Molecular Sciences

Article Title: Cell Shape and Matrix Stiffness Impact Schwann Cell Plasticity via YAP/TAZ and Rho GTPases

doi: 10.3390/ijms22094821

Figure Lengend Snippet: When cell spreading area remains constant, inhibition of RhoA and YAP/TAZ significantly upregulate the expression of c-Jun and p75NTR. Schwann cells (SCs) were seeded into ( A ) 900 μm 2 , ( B ) 1600 μm 2 , or ( C ) 2500 μm 2 micropatterned cell adhesive islands. F-actin (red), nucleus (blue), c-Jun (green), and p75NTR (green) are visualized by immunofluorescence staining. Scale bar = 10 μm. ( D , E ) Statistical analysis shows the expression of c-Jun and p75NTR after Y-27632 and Verteporfin treatment. Mean pixel fluorescent intensities are measured for quantification. For fluorescent intensity quantification of micropatterned SCs, 5 unique trials with a minimum of 51 single micropatterned SCs per trial were quantified. ( F ) Microarray analysis indicates that SC with larger spreading area showed higher RhoA- and YAP/TAZ-associated genes and a decline in SC regenerative markers. Data are presented as the mean ± SEM. * p < 0.05, ** p < 0.005, *** p < 0.0005, p -values found in .

Article Snippet: Y-27632 (Tocris, 1254) (20 μM) was used to inhibit Rho-associated, coiled-coil-containing protein kinase (ROCK) activity, and Verteporfin (Tocris, 5305) (1 μM) was used to inhibit active (nuclear) YAP/TAZ.

Techniques: Inhibition, Expressing, Adhesive, Immunofluorescence, Staining, Microarray

Journal: Developmental cell

Article Title: E2F/DP prevents cell cycle progression in endocycling fatbody cells by suppressing dATM expression

doi: 10.1016/j.devcel.2017.11.008

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit anti- p-S137 H2Av (IF:1/100) , Rockland , 600-401-914.

Techniques: Gene Expression, Microarray, Library Quantification, Single Cell Gel Electrophoresis, Reverse Transcription, SYBR Green Assay, Mass Spectrometry, DNA Sequencing, Control, Sequencing, Luciferase, Software