Journal: PloS one

Article Title: Cancer cells resistant to therapy promote cell surface relocalization of GRP78 which complexes with PI3K and enhances PI(3,4,5)P3 production.

doi: 10.1371/journal.pone.0080071

Figure Lengend Snippet: Figure 3. Cell surface GRP78 forms complex with the PI3K. (A) Confocal microscopy detection of co-localization of endogenous sGRP78 with p85 in C4-2B cells treated with Tg for 8 h. Non-permeabilized cells were first stained with anti-GRP78 antibody (MAb159) to detect sGRP78 (green) (left). The cells were then permeabilized and then stained with anti-p85 antibody (red). (B) Co-localization of cell surface F-GRP78 with p85 in HeLa cells. Non-permeabilized HeLa cells transfected with the F-GRP78 expression vector were first stained with anti-FLAG antibody to detect surface F- GRP78 (green) (left), then permeabilized and stained with anti-p85 antibody (red). In both (A) and (B), the nuclei were stained by DAPI (blue) and the boxed regions (white square) of the compressed Z-stack images (left panels) were enlarged and shown on the right panels in single confocal section planes (thickness 0.38 mm).The merged images showed co-localizations (yellow) of cell surface GRP78 and p85 detected at multiple corresponding sites (white arrows). Scale bars represent 2 mm. (C) Scheme for detection of interaction between cell surface F-GRP78 and p85. Biotinylated cell surface proteins were purified by monomeric avidin column, followed by immunoprecipitation (IP) and Western blot. (D) 293T cells were transfected with F- GRP78 expression vector. Cell surface proteins eluted from the monomeric avidin column (input) as described in (C) were subjected to immunoprecipitation (IP) with either anti-FLAG antibody or isotype IgG serving as negative control. F-GRP78, p85 and p110a levels in immunoprecipitated complex were measured by Western blot with anti-FLAG, anti-p85 and anti-p110a antibodies. doi:10.1371/journal.pone.0080071.g003

Article Snippet: Antibodies used were: mouse anti-FLAG antibody (Sigma-Aldrich), 1:1000; mouse anti-GRP78 antibody MAb159 (gift from P. Gill, USC), 1:2000; rabbit anti-PI3K p85 antibody (#4292, Cell Signaling Technology, Danvers, MA), 1:1000; rabbit anti-PI3K p110a antibody (#4249, Cell Signaling Technology, Danvers, MA), 1:1000; mouse anti-EphB4 antibody (gift from P. Gill, USC), 1:1000; rabbit antiNKA a1 (Na, K-ATPase a1) antibody (Cell Signaling Technology), 1:1000; mouse anti-b-actin antibody (Sigma-Aldrich), 1:5000.

Techniques: Confocal Microscopy, Staining, Transfection, Expressing, Plasmid Preparation, Purification, Avidin-Biotin Assay, Immunoprecipitation, Western Blot, Negative Control

Journal: PLoS Genetics

Article Title: The Wilms Tumor Gene, Wt1 , Is Critical for Mouse Spermatogenesis via Regulation of Sertoli Cell Polarity and Is Associated with Non-Obstructive Azoospermia in Humans

doi: 10.1371/journal.pgen.1003645

Figure Lengend Snippet: Reproductive tracts of control (A) and Wt1 −/flox ; Cre-ER TM (B) males 3 weeks after Tamoxifen-induced Wt1 ablation. Compared to the control mice, the size of Wt1 −/flox ; Cre- ER TM testes is dramatically reduced (C). (D–I) Cross-sections of control and Wt1 −/flox ; Cre- ER TM testes at 1 (D, G), 2 (E, H), and 3 (F, I) weeks after Tamoxifen treatment. Vacuolization was detected in a small number of tubules at 1 week after Tamoxifen induction (G) and severe epithelial vacuolization (arrows) was noted at 2 weeks after Tamoxifen induction in Wt1 −/flox ; Cre- ER TM testis (H). Massive cell loss with empty tubules (asterisks) was observed in Wt1 −/flox ; Cre- ER TM testis at 3 weeks after Tamoxifen treatment (I).

Article Snippet: IHC analysis of tissues from at least three mice for each genotype was performed using a Vectastain ABC (avidin–biotin–peroxidase) kit (Vector Laboratories, Burlingame, CA) as recommended and using antibodies to WT1 (Santa Cruz, sc-192) and GCNA1 (gift of Dr. George Enders).

Techniques: Control

Journal: PLoS Genetics

Article Title: The Wilms Tumor Gene, Wt1 , Is Critical for Mouse Spermatogenesis via Regulation of Sertoli Cell Polarity and Is Associated with Non-Obstructive Azoospermia in Humans

doi: 10.1371/journal.pgen.1003645

Figure Lengend Snippet: In control testes, the seminiferous tubules are filled with GCNA1 positive germ cells (C), whereas, very few GCNA1 positive germ cells (D, black arrows) are noted in the Wt1 −/flox ; Cre- ER TM testes and some tubules are completely void of germ cells (D, asterisks). Wt1 positive Sertoli cells are present in both control (A) and Wt1 −/flox ; Cre- ER TM (B) testes. The cauda epididymes of control mice are filled with mature sperms (E, black arrows); in contrast, only immature spermatocyte and cell debris (arrow heads) are present in the cauda epididymes of Wt1 −/flox ; Cre-ER TM mice and no mature sperm are detectable (F).

Article Snippet: IHC analysis of tissues from at least three mice for each genotype was performed using a Vectastain ABC (avidin–biotin–peroxidase) kit (Vector Laboratories, Burlingame, CA) as recommended and using antibodies to WT1 (Santa Cruz, sc-192) and GCNA1 (gift of Dr. George Enders).

Techniques: Control

Journal: PLoS Genetics

Article Title: The Wilms Tumor Gene, Wt1 , Is Critical for Mouse Spermatogenesis via Regulation of Sertoli Cell Polarity and Is Associated with Non-Obstructive Azoospermia in Humans

doi: 10.1371/journal.pgen.1003645

Figure Lengend Snippet: Electropherogram showing single-nucleotide mutation of WT1 in patients with non-obstructive azoospermia.

Article Snippet: IHC analysis of tissues from at least three mice for each genotype was performed using a Vectastain ABC (avidin–biotin–peroxidase) kit (Vector Laboratories, Burlingame, CA) as recommended and using antibodies to WT1 (Santa Cruz, sc-192) and GCNA1 (gift of Dr. George Enders).

Techniques: Mutagenesis

Journal: PLoS Genetics

Article Title: The Wilms Tumor Gene, Wt1 , Is Critical for Mouse Spermatogenesis via Regulation of Sertoli Cell Polarity and Is Associated with Non-Obstructive Azoospermia in Humans

doi: 10.1371/journal.pgen.1003645

Figure Lengend Snippet: In control testes, biotin tracer is restricted to the testicular interstitium and the basal compartment of the seminiferous tubules, with no tracer observed in the tubular lumen (A). Biotin tracer is detected along the Sertoli cell plasma membranes from the basement membrane to the lumen in some seminiferous tubules (asterisks) of Wt1 −/flox ; Cre- ER TM testes (B). By TEM a normal BTB structure (E) and apical ES ultrastructure with well-organized actin bundles(C, arrows) is observed in control testes, but Wt1 mutant testes display abnormal apical ES structures (D, arrows) and the BTB structure is disrupted with numerous blisters (F, asterisks and arrow heads).

Article Snippet: IHC analysis of tissues from at least three mice for each genotype was performed using a Vectastain ABC (avidin–biotin–peroxidase) kit (Vector Laboratories, Burlingame, CA) as recommended and using antibodies to WT1 (Santa Cruz, sc-192) and GCNA1 (gift of Dr. George Enders).

Techniques: Control, Clinical Proteomics, Membrane, Mutagenesis

Journal: PLoS Genetics

Article Title: The Wilms Tumor Gene, Wt1 , Is Critical for Mouse Spermatogenesis via Regulation of Sertoli Cell Polarity and Is Associated with Non-Obstructive Azoospermia in Humans

doi: 10.1371/journal.pgen.1003645

Figure Lengend Snippet: Control SCs display a typical epithelial morphology (A and E), while Wt1 -ablated SCs display a mesenchyme-like morphology (C and E). The tight junctions between SCs were assessed by ZO-1 staining. ZO-1 protein is present along the boundary between control SCs (B, white arrows). In contrast, ZO-1 protein is diffused in the cytosol and not detected along the cell boundary in Wt1 -deficient SCs (D). (F) FITC-dextran flux assays indicated increased permeability in Wt1 -deficient SCs compared to control SCs. * p <0.05.

Article Snippet: IHC analysis of tissues from at least three mice for each genotype was performed using a Vectastain ABC (avidin–biotin–peroxidase) kit (Vector Laboratories, Burlingame, CA) as recommended and using antibodies to WT1 (Santa Cruz, sc-192) and GCNA1 (gift of Dr. George Enders).

Techniques: Control, Staining, Permeability

Journal: PLoS Genetics

Article Title: The Wilms Tumor Gene, Wt1 , Is Critical for Mouse Spermatogenesis via Regulation of Sertoli Cell Polarity and Is Associated with Non-Obstructive Azoospermia in Humans

doi: 10.1371/journal.pgen.1003645

Figure Lengend Snippet: (A) The expression of cell polarity related genes ( E-cadherin, Par6b, Cdc42bp5, Sfn ) and Wnt signaling genes ( Wnt4 and Wnt11 ) is significantly decreased in Wt1 -deficient SCs (white bars). (B) E-cadherin and Espin protein expression is significantly reduced in Wt1 -deficient SCs, whereas the expression of occludin , N-cadherin, Zo-1 , and β-catenin is not changed. (C) The expression of E-cadherin, Par6b, Wnt4, Wnt11 , and Cdc42ep5 in Wt1 -deficient SCs was rescued by transfection with Wt1 expressing adenovirus. (D) The expression of Par6b was induced by Wnt4 and Wnt11 expressing adenovirus in Wt1 -deficient SCs. (E) The expression of E-cadherin was not rescued by Wnt4 and Wnt11 expressing adenovirus in Wt1 -deficient SCs. (F) Wnt4 was knocked down by two different siRNAs (siWnt4-2 and siWnt4-3) in SCs and the expression of E-cadherin and Par6b was significantly decreased compared to control cells (NC) treated with a scrambled siRNA. * p <0.05.

Article Snippet: IHC analysis of tissues from at least three mice for each genotype was performed using a Vectastain ABC (avidin–biotin–peroxidase) kit (Vector Laboratories, Burlingame, CA) as recommended and using antibodies to WT1 (Santa Cruz, sc-192) and GCNA1 (gift of Dr. George Enders).

Techniques: Expressing, Transfection, Control

Journal: PLoS Genetics

Article Title: The Wilms Tumor Gene, Wt1 , Is Critical for Mouse Spermatogenesis via Regulation of Sertoli Cell Polarity and Is Associated with Non-Obstructive Azoospermia in Humans

doi: 10.1371/journal.pgen.1003645

Figure Lengend Snippet: (A) The expression of Wnt4, Wnt11 , and E-cadherin in Wt1 -deficient SCs was rescued by wild type Wt1 expressing adenovirus, but not Wt1 R362Q and Wt1 K386R expressing adenovirus. Co-transfection of Wt1 R362Q and Wt1 K386R in Wt1 -deficient SCs did not affect Wt1 induced Wnt4, Wnt11 , and E-cadherin expression. (B) Schematic image of Wnt4 promoter, black oval indicated the predicted Wt1 binding site. (C) A 211 bp band in Wnt4 promoter was amplified after pulled down with Wt1 antibody in Wt1 expressing adenovirus transfected HepG2 cells, but not in Wt1 R362Q and Wt1 K386R expressing adenovirus transfected HepG2 cells. Histone3 antibody and input was used as positive control, and IgG and vector was used as negative control.

Article Snippet: IHC analysis of tissues from at least three mice for each genotype was performed using a Vectastain ABC (avidin–biotin–peroxidase) kit (Vector Laboratories, Burlingame, CA) as recommended and using antibodies to WT1 (Santa Cruz, sc-192) and GCNA1 (gift of Dr. George Enders).

Techniques: Expressing, Cotransfection, Binding Assay, Amplification, Transfection, Positive Control, Plasmid Preparation, Negative Control