Journal:

Article Title: Adenovirus-Mediated p21 (WAF1/SDII/CIP1) Gene Transfer Induces Apoptosis of Human Cervical Cancer Cell Lines

doi:

Figure Lengend Snippet: Expression of p21 protein detected by Western blot analysis. The cervical cancer cells, Z172, SiHa, C33A, and HeLa, were infected with sense p21 or antisense p21 adenovirus at a MOI of 25 or with PBS (mock); after 48 h, protein lysates were isolated and analyzed by immunoblotting with anti-p21 antibody (Pharmingen).

Article Snippet: Then the sections were processed as described previously ( 2 ) and incubated with an appropriate dilution of primary antibody against p21 protein (Pharmingen).

Techniques: Expressing, Western Blot, Infection, Isolation

Journal:

Article Title: Adenovirus-Mediated p21 (WAF1/SDII/CIP1) Gene Transfer Induces Apoptosis of Human Cervical Cancer Cell Lines

doi:

Figure Lengend Snippet: In vitro effect of p21 on cell growth. The growth of these four cervical cancer cell lines was determined by counting the cell number at different time points after infection of cells at a MOI of 25 with sense p21 or antisense p21 adenovirus. The results are the mean of three separate experiments.

Article Snippet: Then the sections were processed as described previously ( 2 ) and incubated with an appropriate dilution of primary antibody against p21 protein (Pharmingen).

Techniques: In Vitro, Infection

Journal:

Article Title: Adenovirus-Mediated p21 (WAF1/SDII/CIP1) Gene Transfer Induces Apoptosis of Human Cervical Cancer Cell Lines

doi:

Figure Lengend Snippet: DNA fragmentation assays. Low-molecular-weight DNA was harvested from attached and detached cells infected with sense p21 or antisense p21 adenovirus, and the DNA was analyzed by electrophoresis on a 1.4% agarose gel.

Article Snippet: Then the sections were processed as described previously ( 2 ) and incubated with an appropriate dilution of primary antibody against p21 protein (Pharmingen).

Techniques: Molecular Weight, Infection, Electrophoresis, Agarose Gel Electrophoresis

Journal:

Article Title: Adenovirus-Mediated p21 (WAF1/SDII/CIP1) Gene Transfer Induces Apoptosis of Human Cervical Cancer Cell Lines

doi:

Figure Lengend Snippet: In vitro analysis for apoptosis. Cells infected with sense p21 adenovirus, antisense p21 adenovirus, or PBS only (mock) for 2 days were stained with acridine orange and observed by fluorescence microscopy (a and d) HeLa and C33A cells infected with sense p21 adenovirus, respectively; (b and e) HeLa and C33A cells infected with antisense p21 adenovirus, respectively; (c and f) HeLa and C33A cells without virus infection (mock infected), respectively. The apoptotic bodies are indicated by arrowheads in panels a and d.

Article Snippet: Then the sections were processed as described previously ( 2 ) and incubated with an appropriate dilution of primary antibody against p21 protein (Pharmingen).

Techniques: In Vitro, Infection, Staining, Fluorescence, Microscopy

Journal:

Article Title: Adenovirus-Mediated p21 (WAF1/SDII/CIP1) Gene Transfer Induces Apoptosis of Human Cervical Cancer Cell Lines

doi:

Figure Lengend Snippet: p21-mediated tumor growth inhibition. HeLa and SiHa cells (2.5 × 106) were separately injected subcutaneously into the flanks of nude mice. When the tumors grew to about 75 mm3, the animals were reanesthetized and 2.5 × 107 PFU of adenovirus encoding sense p21 or antisense p21 or PBS only (mock infection) was injected into the tumor in five nude mice in each cell line group. The volumes of the tumors in five nude mice in each group were measured weekly. The error bars indicate the standard deviations of tumor volumes of five mice.

Article Snippet: Then the sections were processed as described previously ( 2 ) and incubated with an appropriate dilution of primary antibody against p21 protein (Pharmingen).

Techniques: Inhibition, Injection, Infection

Journal:

Article Title: Adenovirus-Mediated p21 (WAF1/SDII/CIP1) Gene Transfer Induces Apoptosis of Human Cervical Cancer Cell Lines

doi:

Figure Lengend Snippet: In vivo analysis of apoptosis by an in situ TUNEL assay. In situ end-labeling analysis was performed on paraffin-embedded sections obtained from tumor-bearing nude mice which were injected with sense p21 or antisense p21 adenovirus or PBS only (mock infected) 3 days earlier. SiHa cells were injected with PBS only (a and e), antisense p21 adenovirus (b and f), or sense p21 adenovirus (c, d, g, and h). (a to d) Hematoxylin and eosin stain; (e to h) fluorescent stain of apoptotic cells as shown by the in situ TUNEL assay. Magnifications, ×40 (a, b, c, e, f, and g) and ×300 (d and h); panels d and h are magnifications of panels c and g, respectively.

Article Snippet: Then the sections were processed as described previously ( 2 ) and incubated with an appropriate dilution of primary antibody against p21 protein (Pharmingen).

Techniques: In Vivo, In Situ, TUNEL Assay, End Labeling, Injection, Infection, H&E Stain, Staining

Journal:

Article Title: Adenovirus-Mediated p21 (WAF1/SDII/CIP1) Gene Transfer Induces Apoptosis of Human Cervical Cancer Cell Lines

doi:

Figure Lengend Snippet: Expression of p21 in tumor tissues. At 36 h postinfection, the paraffin-embedded sections were obtained from sense p21 virus-treated (a), antisense p21 virus-treated (b) or mock-treated (c) tumors and stained with p21 antibody (Pharmingen) by the immunohistochemistry assay. Hematoxylin was used for counterstaining.

Article Snippet: Then the sections were processed as described previously ( 2 ) and incubated with an appropriate dilution of primary antibody against p21 protein (Pharmingen).

Techniques: Expressing, Staining, Immunohistochemistry

Journal:

Article Title: Adenovirus-Mediated p21 (WAF1/SDII/CIP1) Gene Transfer Induces Apoptosis of Human Cervical Cancer Cell Lines

doi:

Figure Lengend Snippet: Bcl-2 cannot protect against p21-mediated apoptosis in cervical cancer cells. (A) Expression of Bcl-2 protein. Cellular proteins were extracted, then separated by a SDS-PAGE, and subjected to immunoblot analysis with antibody recognizing Bcl-2 (Santa Cruz). Lanes 1 and 2 represent SiHa cells and SiHa/bcl2 cells (which contain the bcl-2 gene). (B) Low-molecular-weight DNA was harvested from attached and detached SiHa/bcl2 cells which were infected with sense p21 (lane 2) or antisense p21 (lane 1) adenovirus and then analyzed by electrophoresis on a 1.4% agarose gel. (C) SiHa cells with or without Bcl-2 protein infected with sense p21 or antisense p21 adenovirus for 2 days were stained with acridine orange and observed with a fluorescence microscope. Panels a and b represent SiHa cells infected with sense p21 and antisense p21 adenovirus, respectively. Panels c and d represent SiHa/bcl2 cells infected with sense p21 and antisense p21 adenovirus, respectively. The apoptotic cells were observed in panels a and c (arrowheads).

Article Snippet: Then the sections were processed as described previously ( 2 ) and incubated with an appropriate dilution of primary antibody against p21 protein (Pharmingen).

Techniques: Expressing, SDS Page, Western Blot, Molecular Weight, Infection, Electrophoresis, Agarose Gel Electrophoresis, Staining, Fluorescence, Microscopy

Journal: Molecular Medicine

Article Title: Gene Expression in Temporal Lobe Epilepsy is Consistent with Increased Release of Glutamate by Astrocytes

doi: 10.2119/2006-00079.Lee

Figure Lengend Snippet: Gene Expression Fold Changes *

Article Snippet: Immunohistochemistry An approximately 3 mm thick slice of tissue adjacent to the one used for DNA microarray studies was fixed by immersion in a 4% paraformaldehyde solution in phosphate buffer (pH 7.4) for 6–8 h Vibratome sections (50 μm thick) were immunostained according to the Avidin-Biotin Complex method ( 31 ) with primary antibodies against AQP4 (1:10,000, Chemicon, Temecula, CA), DMN (1:5000, Novocastra, Newcastle, UK), CD44 (1:500, RDI, Flanders, NJ), PLEC1 (1:500, BD Transduction Laboratories) and CXCR4 (1:1000, ProSci Inc., Poway, CA) using procedures previously described ( 18 ).

Techniques: Expressing

Journal: Molecular Medicine

Article Title: Gene Expression in Temporal Lobe Epilepsy is Consistent with Increased Release of Glutamate by Astrocytes

doi: 10.2119/2006-00079.Lee

Figure Lengend Snippet: This figure shows photomicrographs of the localization in area CA1 pyramidal layer of non-sclerotic (non-MTLE, A, C. E. G, I) and sclerotic (MTLE, B, D, F, H, J) hippocampi of some proteins whose genes showed changes in expression in this study. A & B show the immunolocalization of AQP4 (Aquaporin 4). Note the reduction in immunoreactivity in astrocytic foot processes around blood vessels in MTLE and increase in astrocytic processes throughout the pyramidal layer. C & D show reduction in DMD (Dystrophin) around blood vessels in MTLE. E & F show localization of CD44 (CD44 antigen). In the non-sclerotic hippocampus (E) immunoreactivity is seen on fiber-like processes (arrow) extending into the pyramidal layer from the stratum oriens. Scattered among these fibers are CD44 positive astrocyte cell bodies (not shown). In MTLE, many more astrocytes are immunoreactive and form a dense network throughout the region with more intense staining in perivascular foot processes (arrow in F). G & H show PLEC1 (Pectin 1, intermediate filament binding protein 500kDa) immunoreactivity, which is increased in expression in astrocyte cell bodies and perivascular foot processes (arrow). I & J show an increase in CXCR4 (Chemokine (C-X-C motif) receptor 4) expression on microglia in the sclerotic hippocampus (arrow). CXCR4 is increased on a small proportion of astrocytes as well.

Article Snippet: Immunohistochemistry An approximately 3 mm thick slice of tissue adjacent to the one used for DNA microarray studies was fixed by immersion in a 4% paraformaldehyde solution in phosphate buffer (pH 7.4) for 6–8 h Vibratome sections (50 μm thick) were immunostained according to the Avidin-Biotin Complex method ( 31 ) with primary antibodies against AQP4 (1:10,000, Chemicon, Temecula, CA), DMN (1:5000, Novocastra, Newcastle, UK), CD44 (1:500, RDI, Flanders, NJ), PLEC1 (1:500, BD Transduction Laboratories) and CXCR4 (1:1000, ProSci Inc., Poway, CA) using procedures previously described ( 18 ).

Techniques: Expressing, Staining, Binding Assay

Journal: Molecular Oncology

Article Title: Growth arrest‐specific protein 2 ( GAS2 ) interacts with CXCR4 to promote T‐cell leukemogenesis partially via c‐MYC

doi: 10.1002/1878-0261.13306

Figure Lengend Snippet: GAS2 interacts with CXCR4. (A) The expression of CXCR4 in Calpain2 silenced (shCPN2) and control (Scrambled) Jurkat cells was analyzed by western blot ( n = 3). (B) The protein extraction of Jurkat cells was prepared. An aliquot of the extract was used as an untreated control. One aliquot of the extract was supplied with Ca ++ only, and the other aliquot was added with both Ca ++ and Calpain inhibitor (Z‐LLY‐FMK); both aliquots were incubated at 37 °C for 8 h. Finally, the three aliquots of lysates were separated with SDS/PAGE and transferred onto a PVDG membrane. Ponceau S staining was used to monitor the loading of the samples. The expression of CXCR4 and GAPDH was analyzed by western blot. ( n = 4) (C) The dominant negative form of GAS2 (GAS2DN) was overexpressed in Jurkat cells, the expression of CXCR4 in these cells together with their control was analyzed by western blot ( n = 3). (D) Calpain2 was silenced in GAS2DN overexpressed Jurkat cells, and then CXCR4 expression in these cells together with the control cells was analyzed by western blot ( n = 3). (E) Flag‐tagged GAS2 and/or HA tagged CXCR4 were overexpressed in 293 T cells, and coimmunoprecipitation (Co‐IP) experiment was performed to analyze the interaction between GAS2 and CXCR4 ( n = 2). (F) Immunoprecipitation (IP) against GAS2 in Jurkat cells was performed (left panel), and the expression of CXCR4 and Calpain2 (CPN2) was detected with immunoblot (IB). Conversely, IP against CXCR4 in Jurkat cells was conducted (right panel), and the expression of GAS2 and CPN2 was detected with IB ( n = 2). (G) The expression of both GAS2 (green) and CXCR4 (red) was analyzed with confocal microscopy ( n = 2). (H) Additionally, the expression of both Calpain2 (green) and CXCR4 (red) was analyzed with confocal microscopy ( n = 2). The scale bar equals 5 or 20 μm as indicated in each graph.

Article Snippet: Briefly, the cells were first incubated with antibody against GAS2 (ab109762; Abcam, Waltham, MA, USA) or Calpain2 (A4066; ABclonal, Wuhan, China), and they were incubated with antibody against CXCR4 (60042‐1‐Ig; Proteintech).

Techniques: Expressing, Western Blot, Protein Extraction, Incubation, SDS Page, Staining, Dominant Negative Mutation, Co-Immunoprecipitation Assay, Immunoprecipitation, Confocal Microscopy