Journal: The Journal of clinical endocrinology and metabolism

Article Title: Coexpression of fractalkine and its receptor in normal human endometrium and in endometrium from users of progestin-only contraception supports a role for fractalkine in leukocyte recruitment and endometrial remodeling.

doi: 10.1210/jc.2003-031379

Figure Lengend Snippet: FIG. 1. Immunohistochemical localization of fractalkine (A–H and R–T) in human endometrium. A, Proliferative phase endometrium showing diffuse immunostaining for fractalkine in glandular epithelium (GE) and leukocytes (). More intense staining was seen in the early secretory (B), midsecretory (C), and late secretory (D and E) phases. Staining appeared to be in vesicles (V) in the basal (ba) region in GE in the early secretory phase (B), more diffuse in the midsecretory phase (C), and apically located (ap) in the late secretory phase (D and inset), when strong staining was also observed in blood vessels (bv) and decidualizing stroma (Dec) surrounding the spiral arterioles (E). Fractalkine was also identified in decidua (Dec) in early pregnancy (F). No staining was observed after preadsorption of antibody with fractalkine peptide (E and inset). Fractalkine-staining leukocytes were identified by colocalization studies on adjacent 2-m sections. Staining pairs are fractalkine (G)

Article Snippet: Negative controls were also included, where the primary antibody was preabsorbed with 1 g/ml fractalkine blocking peptide (SC-7226, Santa Cruz Biotechnology) at 4 C for 48 h. A section from a single block of endometrial tissue was included in each staining run for quality control.

Techniques: Immunohistochemical staining, Immunostaining, Staining

Journal: The Journal of clinical endocrinology and metabolism

Article Title: Coexpression of fractalkine and its receptor in normal human endometrium and in endometrium from users of progestin-only contraception supports a role for fractalkine in leukocyte recruitment and endometrial remodeling.

doi: 10.1210/jc.2003-031379

Figure Lengend Snippet: FIG. 2. Fractalkine (FKN) immunostaining intensity in human en- dometrium, across the menstrual cycle and in early pregnancy. A, Semiquantitative scoring of FKN in glandular epithelium and stro- mal cells in the following tissues: menstrual (ME; n 5), proliferative (PR; n 7), early secretory (ES; n 6), midsecretory (MS; n 6), late secretory (LS; n 6), and early pregnancy (PREG; n 10). Results shown are the mean SEM. *, P 0.05 (in ES, LS, and PREG, glandular immunostaining levels are significantly elevated compared PR and MS levels). ††, P 0.01 (in LS, stromal immunostaining levels are significantly elevated compared with MS levels). B, Semiquan- titative scoring of FKN in leukocytes. Results shown are the mean SEM.

Article Snippet: Negative controls were also included, where the primary antibody was preabsorbed with 1 g/ml fractalkine blocking peptide (SC-7226, Santa Cruz Biotechnology) at 4 C for 48 h. A section from a single block of endometrial tissue was included in each staining run for quality control.

Techniques: Immunostaining

Journal: The Journal of clinical endocrinology and metabolism

Article Title: Coexpression of fractalkine and its receptor in normal human endometrium and in endometrium from users of progestin-only contraception supports a role for fractalkine in leukocyte recruitment and endometrial remodeling.

doi: 10.1210/jc.2003-031379

Figure Lengend Snippet: FIG. 4. Semiquantitative scoring of glandular epithelium, stromal, and leukocyte immunostaining levels for fractalkine (FKN) and CX3CR1 in human endometrium exposed to P-only contraceptives. A, FKN localization in endometrium before insertion of LNG-IUS in the proliferative phase (PR; n 4) or secretory phase (SEC; n 5) and at 3, 6, and 12 months postinsertion (n 3/group). *, P 0.05, significant increase in FKN-immunopositive leukocytes 3 months postinsertion compared with preinsertion controls. Epithelial immu- nostaining was absent 3 months postinsertion and significantly de- creased 12 months postinsertion (*, P 0.05). Stromal cells were strongly positive for FKN at all stages postinsertion (*, P 0.05). B, FKN immunostaining in endometrium from users of Depo-Provera (DEPO) and Norplant (NORP) compared with nonusers (controls) from proliferative phase (PR; n 7) and secretory phase (SEC; n

Article Snippet: Negative controls were also included, where the primary antibody was preabsorbed with 1 g/ml fractalkine blocking peptide (SC-7226, Santa Cruz Biotechnology) at 4 C for 48 h. A section from a single block of endometrial tissue was included in each staining run for quality control.

Techniques: Immunostaining

Journal: The Journal of clinical endocrinology and metabolism

Article Title: Coexpression of fractalkine and its receptor in normal human endometrium and in endometrium from users of progestin-only contraception supports a role for fractalkine in leukocyte recruitment and endometrial remodeling.

doi: 10.1210/jc.2003-031379

Figure Lengend Snippet: FIG. 5. Diagrammatic illustration of fractalkine expression and its potential functional significance. Epithelial (E) and stromal (S) fractalkine (FKN) levels are maximal during the secretory phase of the menstrual cycle. In the midsecretory (MS) phase, there is a dramatic influx of macrophages; FKN produced by E and S could be involved in their positioning and activation. This also coincides with the time of blastocyst implantation; FKN could be acting to increase blastocyst/epithelium receptivity. Production of FKN by endometrial vasculature only occurs premenstrually. Macrophages and neutrophils express CX3CR1; therefore, endometrial FKN could be involved in their selective recruitment, positioning, and activation for the initiation of menstruation. Prolif, Proliferative phase; LS, late secretory phase; V, fractalkine positive vessel staining.

Article Snippet: Negative controls were also included, where the primary antibody was preabsorbed with 1 g/ml fractalkine blocking peptide (SC-7226, Santa Cruz Biotechnology) at 4 C for 48 h. A section from a single block of endometrial tissue was included in each staining run for quality control.

Techniques: Expressing, Functional Assay, Produced, Activation Assay, Staining

Journal: The Journal of clinical endocrinology and metabolism

Article Title: Cytoplasmic expression of fibroblast growth factor receptor-4 in human pituitary adenomas: relation to tumor type, size, proliferation, and invasiveness.

doi: 10.1210/jc.2003-031489

Figure Lengend Snippet: FIG. 1. Detection of FGFR4 reactivity in pituitary cells. A–D, Immunohistochem- ical localization of FGFR4 in human pi- tuitary adenomas. C-terminal FGFR4 immunoreactivity is seen in the cyto- plasm of tumor cells in varied tumor types. A, GH cell adenoma. B, ACTH cell adenoma. C, FSH/LH adenoma. D, Null cell adenoma. The immunoreactivity of FGFR4 is variable but always very in- tense throughout the cytoplasm (original magnification 50–100). E and F, Im- munohistochemical localization of FGFR4 and ptd-FGFR4 in transfected pituitary cells. Rat pituitary tumor-derived GH4 cells were stably transfected with full- length FGFR4 (E) or ptd-FGFR4 (F). Im- munocytochemical examination was per- formed using an antibody that recognizes the C terminus of FGFR4. Note the pre- dominant cytoplasmic pattern of staining in ptd-FGFR4 transfected cells, com- pared with the membrane reactivity in wild-type FGFR4-transfected cells. G, Western blot detection of FGFR4 in hu- man pituitary adenomas. Protein lysates from seven human gonadotroph adeno- mas were electrophoresed and blotted with an antibody that recognizes the C terminus of FGFR4. The extreme right lane contains a positive control from HEK293 cells transiently transfected with ptd-FGFR4 (upper panel). Preab- sorption of the primary antibody with pu- rified antigen abolishes the lower 65-kDa protein (lower panel). The band migrat- ing just above 65 kDa is a nonspecific species that is not abolished by preab- sorption.

Article Snippet: Furthermore, the specificity of all reactions for FGFR4 was verified by replacing the primary antibody with normal serum, examining negative control tissues, and preabsorbing primary antibody with purified FGFR4 peptide (Santa Cruz, antibody: peptide/6:1).

Techniques: Transfection, Derivative Assay, Stable Transfection, Staining, Membrane, Western Blot, Positive Control

Journal: The Journal of clinical endocrinology and metabolism

Article Title: Cytoplasmic expression of fibroblast growth factor receptor-4 in human pituitary adenomas: relation to tumor type, size, proliferation, and invasiveness.

doi: 10.1210/jc.2003-031489

Figure Lengend Snippet: FIG. 2. Cytoplasmic expression levels of FGFR4 and Ki-67 LI in pituitary ad- enomas. A, The expression levels of FGFR4 in macroadenomas are signifi- cantly higher than those in microadeno- mas (P 0.02). B, The expression level of FGFR4 in invasive adenomas is not significantly different from noninvasive adenomas. C, The mean Ki-67 LI (per- cent) was significantly higher in tumors that express high levels of FGFR4 than in the low FGFR4-expressing group, which in turn was higher than in the FGFR4-negative group of tumors (P 0.03; P 0.002, respectively). D and E, Ki-67 LI was higher in macroadenomas and invasive adenomas than in mi- croadenomas and noninvasive adeno- mas (P 0.05; P 0.01, respectively).

Article Snippet: Furthermore, the specificity of all reactions for FGFR4 was verified by replacing the primary antibody with normal serum, examining negative control tissues, and preabsorbing primary antibody with purified FGFR4 peptide (Santa Cruz, antibody: peptide/6:1).

Techniques: Expressing

Journal: The Journal of Cell Biology

Article Title: PKCζ mediates disturbed flow-induced endothelial apoptosis via p53 SUMOylation

doi: 10.1083/jcb.201010051

Figure Lengend Snippet: PKCζ mediates ONOO − -induced p53 nuclear export and p53–Bcl-2 binding instead of the regulation of p53 transcriptional activity. (A and B) HUVECs were transfected with the p53-Luc reporter and Renilla luciferase–encoding plasmid (pRL- thymidine kinase) used as an internal control reporter together with p53–wild type or vector alone (pcDNA3.1; A). Some cells were further transfected with or without pcDNA3.1-CATζ (B). Transcriptional activity was determined by a reporter plasmid encoding 13 copies of the p53-binding sequence (p53-Luc reporter; ). After 24 h of transfection, p53 transcriptional activity was assayed using the dual-luciferase kit (B), or the cells were further treated with 10 or 50 µM ONOO − for 8 h as indicated, and luciferase activity was assayed (A). Data are representative of triplicates using two or more different preparations of ECs. *, P < 0.05; **, P < 0.01. (C) HUVECs were transduced with Ad-DN-PKCζ or Ad-LacZ as a control for 24 h, treated with vehicle or 100 µM ONOO − for 4 h, and immunostained with anti-p53 followed by DAPI counter staining for nuclei. Bar, 5 µm. (D, top) HUVECs were transduced with Ad-DN-PKCζ or Ad-LacZ for 24 h and stimulated with 100 µM ONOO – for the indicated times. p53–Bcl-2 binding was determined by coimmunoprecipitation with anti-p53 followed by immunoblotting with anti–Bcl-2. p53, PKCζ, and Bcl-2 in total cell lysates were detected by Western blotting with each specific antibody. (bottom) Quantification of p53–Bcl-2 binding expressed as the relative band intensity ratio between coimmunoprecipitated versus total Bcl-2. Results were normalized as described in . n = 3. *, P < 0.05 and **, P < 0.01 compared with the vehicle control, and # , P < 0.05 and ## , P < 0.01 compared with the LacZ control at each time point. Molecular masses are given in kilodaltons. Error bars indicate means ± SD. IB, immunoblot. IP, immunoprecipitation.

Article Snippet: Specificity of anti-p53 staining was tested by staining with anti-p53 preabsorbed with recombinant p53 (Santa Cruz Biotechnology, Inc.) as shown in Fig. S4.

Techniques: Binding Assay, Activity Assay, Transfection, Luciferase, Plasmid Preparation, Control, Sequencing, Transduction, Staining, Western Blot, Immunoprecipitation

Journal: The Journal of Cell Biology

Article Title: PKCζ mediates disturbed flow-induced endothelial apoptosis via p53 SUMOylation

doi: 10.1083/jcb.201010051

Figure Lengend Snippet: PKCζ mediates d-flow–induced p53 SUMOylation and p53–Bcl-2 binding. (A) HeLa cells were transfected for 24 h as indicated with Flag-tagged p53, HA-tagged SUMO3, and HA-tagged CATζ. p53 SUMOylation was detected by immunoprecipitation with anti-Flag followed by Western blotting with anti-SUMO2/3 (top). Both protein expression and immunoprecipitated p53 were confirmed by anti-Flag antibody, and CATζ and SUMO expression were detected with anti-HA. Mono-SUMOylation band (∼74 kD) and poly-SUMOylation bands (>78 kD) were detected. The asterisk indicates mono-SUMOylation of p52. (B) HUVECs were transfected for 24 h with either PIASy or control siRNA as indicated, and then the cells were transfected with HA-CATζ or vector alone for another 24 h. (top) p53 SUMOylation was detected by immunoprecipitation with anti-p53 followed by Western blotting with anti-SUMO2/3. PIASy expression was confirmed by immunoblotting with anti-PIASy, and p53, HA-CATζ, and SUMO expression was confirmed with anti-p53, -HA, and -SUMO2/3, respectively. (C) HUVECs were transfected with either p53 or control siRNA as indicated for 24 h, and then the cells were transduced with an Ad-SENP2 or LacZ with a control for another 24 h. p53 SUMOylation, expression of p53, SENP2, and SUMO were determined as described in Materials and methods. The asterisks indicate nonspecific bands. (D) HUVECs were transduced with Ad-DN-PKCζ or Ad-LacZ as a control for 24 h and then stimulated with d-flow for the indicated times. p53 SUMOylation and p53–Bcl-2 binding were determined as described in Materials and methods. (left graph) Intensities of SUMOylated p53 bands at 74, 82, 130, and 185 kD were quantified by densitometry after subtracting background gel density. After normalization of each control as described in , results were expressed relative to the SUMOylation level in static condition (0 min; 100%). Shown are means ± SD ( n = 3). **, P < 0.01 compared with the vehicle control or the LacZ control at each time point. (E) HUVECs were transfected with either PKCζ or control siRNA as indicated for 24 h and then stimulated with d-flow for 3 h. p53 SUMOylation, p53–Bcl-2 binding, expression of p53, Bcl-2, SUMO, and various PKC isoforms as indicated were determined as described in Materials and methods. Immunoblots are representative of three separate experiments. Molecular masses are given in kilodaltons. IB, immunoblot. IP, immunoprecipitation. KR, K386R. WT, wild type.

Article Snippet: Specificity of anti-p53 staining was tested by staining with anti-p53 preabsorbed with recombinant p53 (Santa Cruz Biotechnology, Inc.) as shown in Fig. S4.

Techniques: Binding Assay, Transfection, Immunoprecipitation, Western Blot, Expressing, Control, Plasmid Preparation, Transduction

Journal: The Journal of Cell Biology

Article Title: PKCζ mediates disturbed flow-induced endothelial apoptosis via p53 SUMOylation

doi: 10.1083/jcb.201010051

Figure Lengend Snippet: ONOO − mediates d-flow–induced PKCζ activation, p53 SUMOylation, and EC apoptosis. (A) ONOO − mediates d-flow–induced PKCζ activation and p53 SUMOylation. HUVECs were pretreated by 5 µM ebselen, 20 µM L-NAME, and 10 µM Mn-TBAP for 30 min and exposed to d-flow for 3 h. PKCζ phosphorylation at Thr560 and p53 SUMOylation were determined as described in Materials and methods. (B and C) Densitometry analyses of p53 SUMOylation (B) and PKCζ phosphorylation (C) were performed as described in . **, P < 0.01 compared with the vehicle control in static condition, and # , P < 0.01 compared with the vehicle control in d-flow stimulation for 3 h. (D and E) HUVECs were pretreated by each inhibitor for 30 min and exposed to d-flow for 36 h followed by TUNEL staining as described in Materials and methods (D), and quantification of apoptosis is shown as the percentage of TUNEL-positive cells (E). Bars, 30 µm. Data are from three separate experiments using two or more different EC preparations (**, P < 0.01 compared with the vehicle control in static condition, and # , P<0.05 and ## , P<0.01 compared with the vehicle control in d-flow stimulation for 36 h). Error bars show means ± SD. Molecular masses are given in kilodaltons. IB, immunoblot. IP, immunoprecipitation.

Article Snippet: Specificity of anti-p53 staining was tested by staining with anti-p53 preabsorbed with recombinant p53 (Santa Cruz Biotechnology, Inc.) as shown in Fig. S4.

Techniques: Activation Assay, Phospho-proteomics, Control, TUNEL Assay, Staining, Western Blot, Immunoprecipitation

Journal: The Journal of Cell Biology

Article Title: PKCζ mediates disturbed flow-induced endothelial apoptosis via p53 SUMOylation

doi: 10.1083/jcb.201010051

Figure Lengend Snippet: ONOO − induces p53 SUMOylation and p53–Bcl-2 binding via PIASy activation. (A and B) HUVECs were transfected with PIASy siRNA (si-PIASy) or control siRNA for 48 h and then stimulated with 100 µM ONOO – for the indicated times. p53 SUMOylation (A) and p53–Bcl-2 binding (B) were determined as described in Materials and methods. (left) PIASy and p53 expressions were detected by Western blotting with appropriate specific antibodies. Densitometric analyses of p53 SUMOylation (A) and p53–Bcl-2 binding (B) were performed as described in . (C) HUVECs were transfected with PIASy or control siRNA for 48 h. After treatment with 100 µM ONOO − for 8 h, apoptotic nuclei were detected by TUNEL staining. Data are expressed as mean percentages ± SD from three independent experiments. *, P < 0.05; **, P < 0.01. Molecular masses are given in kilodaltons. IB, immunoblot. IP, immunoprecipitation.

Article Snippet: Specificity of anti-p53 staining was tested by staining with anti-p53 preabsorbed with recombinant p53 (Santa Cruz Biotechnology, Inc.) as shown in Fig. S4.

Techniques: Binding Assay, Activation Assay, Transfection, Control, Western Blot, TUNEL Assay, Staining, Immunoprecipitation

Journal: The Journal of Cell Biology

Article Title: PKCζ mediates disturbed flow-induced endothelial apoptosis via p53 SUMOylation

doi: 10.1083/jcb.201010051

Figure Lengend Snippet: D-flow induces p53 SUMOylation and apoptosis via PIASy activation. (A) HUVECs were transfected with PIASy siRNA (si-PIASy) or control siRNA for 48 h and then stimulated with d-flow for the indicated times. p53 SUMOylation, expression of PIASy, p53, and SUMO2/3 were detected as described in Material and methods. Densitometric analyses of p53 SUMOylation were performed as described in . (B and C) HUVECs were transfected with PIASy or control siRNA for 48 h. After treatment with d-flow for 36 h, apoptotic nuclei were detected by TUNEL staining (B, bottom), and Western blotting with anti–cleaved caspase 3 (C, top) was performed. Immunoblots of PIASy conformed depletion of PIASy by the specific siRNA (B, top). Densitometry analysis of cleaved caspase 3 expression was performed as described in (bottom). The experiments were performed in triplicate using three different batches of d-flow–stimulated HUVECs. (D) HUVECs were transduced with an adenovirus vector containing p53, p53-K386R (KR; sumoylation defect mutant), or p53-ΔNES (L348,350A; NES mutant) for 24 h and then stimulated with d-flow for 36 h followed by TUNEL staining as described in Materials and methods. (E, top) Quantification of apoptosis shown as the percentage of TUNEL-positive cells. Bars, 30 µm. (bottom) Equal expressions of p53, p53-K386R, and p53-ΔNES were analyzed by Western blotting in ECs. Data are from three separate experiments using two or more different EC preparations. Error bars show means ± SD; *, P < 0.05; **, P < 0.01. Molecular masses are given in kilodaltons. IB, immunoblot. IP, immunoprecipitation. WT, wild type.

Article Snippet: Specificity of anti-p53 staining was tested by staining with anti-p53 preabsorbed with recombinant p53 (Santa Cruz Biotechnology, Inc.) as shown in Fig. S4.

Techniques: Activation Assay, Transfection, Control, Expressing, TUNEL Assay, Staining, Western Blot, Transduction, Plasmid Preparation, Mutagenesis, Immunoprecipitation

Journal: The Journal of Cell Biology

Article Title: PKCζ mediates disturbed flow-induced endothelial apoptosis via p53 SUMOylation

doi: 10.1083/jcb.201010051

Figure Lengend Snippet: PKCζ–PIASy association is critical for p53 SUMOylation and p53–Bcl-2 binding. (A) HUVECs were stimulated with 100 µM ONOO – for the indicated times and subjected to immunoprecipitation with anti-PIASy followed by Western blotting with anti-PKCζ (top). (B and C) Association between PKCζ and PIASy was tested by a mammalian two-hybrid assay. HeLa cells were transfected with plasmids containing Gal4-PKCζ wild type and VP16-PIASy (B) or truncated mutants of VP16-PIASy (C) as well as the Gal4-responsive luciferase reporter pG5-luc. After 24 h of transfection, cells were stimulated with 100 µM ONOO − or vehicle for 16 h, and luciferase activity was quantified. Luciferase activity was normalized with the Renilla luciferase (Luc.) activity . Data are representative of three experiments using two or more different preparations of ECs (means ± SD; **, P < 0.01). (D) PIASy binding to PKCζ occurs via a domain consisting of aa 301–410 of PIASy. HeLa cells were transfected with each of the Flag-tagged PIASy fragments, and then pull-down assays were preformed using anti-Flag and IgG Sepharose beads in the presence of GST-fused recombinant PKCζ. Association of PIASy fragments with GST-PKCζ was assayed by Western blotting with anti-PKCζ. (bottom) PIASy fragment expression was detected by Western blotting with anti-Flag. (E) PIASy Fr3, but not Fr4, inhibited PKCζ–PIASy association. HUVECs were cotransfected with HA-tagged PKCζ wild type, Myc-tagged PIASy wild type, and Flag-tagged PIASy Fr3 or Fr4 for 24 h. Myc-PIASy wild type was immunoprecipitated with anti-Myc followed by immunoblotting with anti-HA (top). The expression of PKCζ, PIASy, and PIASy fragments was detected by Western blotting with specific antibodies. Data are representative of three independent experiments. (F) HUVECs were transfected with Flag-tagged PIASy Fr3 or Fr4 or control vectors for 24 h and then stimulated by d-flow for 3 h. p53 was immunoprecipitated using anti-p53, and d-flow–induced p53 SUMOylation was analyzed by immunoblotting with anti-SUMO2/3 (top). The expression of p53, SUMO, and PIASy fragments was detected by Western blotting with specific antibodies. Data are representative of three independent experiments. Molecular masses are given in kilodaltons. IB, immunoblot. IP, immunoprecipitation. WT, wild type.

Article Snippet: Specificity of anti-p53 staining was tested by staining with anti-p53 preabsorbed with recombinant p53 (Santa Cruz Biotechnology, Inc.) as shown in Fig. S4.

Techniques: Binding Assay, Immunoprecipitation, Western Blot, Two Hybrid Assay, Transfection, Luciferase, Activity Assay, Recombinant, Expressing, Control

Journal: The Journal of Cell Biology

Article Title: PKCζ mediates disturbed flow-induced endothelial apoptosis via p53 SUMOylation

doi: 10.1083/jcb.201010051

Figure Lengend Snippet: D-flow–induced PKCζ–PIASy association in nuclei and p53–Bcl-2 binding in the cytosol. (A and B) HUVECs were stimulated with either static or d-flow for 3 h and immunoassayed with antibodies of mouse anti-PKCζ and rabbit anti-PIASy (A) or mouse anti-p53 and rabbit anti–Bcl-2 (B). After d-flow stimulation, yellow in the merged images represent colocalization between PKCζ and PIASy in nuclei or p53 and Bcl-2 in cytosol. Images were recorded using a confocal microscope equipped with a Plapon 60× 1.42 NA oil lens objective. Shown are representative images from cells analyzed from three independent experiments in which ≥30 cells were analyzed per experiment. Bars, 10 µm.

Article Snippet: Specificity of anti-p53 staining was tested by staining with anti-p53 preabsorbed with recombinant p53 (Santa Cruz Biotechnology, Inc.) as shown in Fig. S4.

Techniques: Binding Assay, Microscopy

Journal: The Journal of Cell Biology

Article Title: PKCζ mediates disturbed flow-induced endothelial apoptosis via p53 SUMOylation

doi: 10.1083/jcb.201010051

Figure Lengend Snippet: Increases in phosphorylated and total PKCζ and nonnuclear p53 expression within the d-flow regions (HP areas) and decreased apoptosis in ECs of p53 −/− mice. (A) A representative epifluorescence image of the whole specimen. Fixed aortas of wild-type mice were cut longitudinally, and the arch region was further cut into two halves. Areas of d-flow (HP area; lesser curvature) are outlined in red, and neighboring areas of s-flow (LP area) are lined in blue. a, artery. (B and C) En face preparations were double stained with anti–VE-cadherin (VE-cad; used as an EC marker) and an anti–total PKCζ antibody (B) or phospho-PKCζ T560 antibody (C). X-y axis images were collected at 0.5-µm increments so that a z stack of ∼4-µm thickness from the luminal surface was obtained. From each image background, fluorescence intensity was subtracted, and the pixel number of the stained region per unit area of the endothelium in HP and LP area within the aortic arch was determined ( n = 3). Areas of d-flow (HP areas; lesser curvature) show both increased total and phospho-PKCζ expression compared with the neighboring areas of s-flow (LP area). Bars, 20 µm. Bar graphs show quantification of total (B) and phospho (C)-PKCζ in HP and LP areas. Data are shown as means ± SEM; *, P < 0.05. (D and E) Increased cytoplasmic p53 localization in HP area ECs. Aortic arches were immunostained for endothelial p53 (green). Nuclei were stained using TO-PRO3 (red). From initial stacked x-y axis images (top), a narrow rectangular area crossing an EC was selected for x-y-z scanning at 0.1-µm increments. Images below the clipped images show rectangular z-axis images. Two representative sets of images (i and ii) are shown for LP (D) and HP (E) areas. Bars, 10 µm. (F) Quantification of nuclear p53. The pixel number of nuclear and nonnuclear regions per cell was determined, and the ratio of nuclear/total intensity was calculated from 60 cells from each HP and LP area (four cells/field, five fields/mouse, and a total of three mice). (G) The number of d-flow–mediated annexin V–positive cells (red) in the HP area is decreased in the mice deficient for p53 (p53 knockout) compared with the wild-type mouse aorta. Anti–VE-cadherin staining (green) was used as a marker for ECs. Bars, 100 µm. (H, left) Quantification of apoptotic cells. Percentages of annexin V–positive cells in LP and HP areas determined from 7-wk wild-type and p53-deficient mice ( n = 3 each) are shown. In HP area, the number of annexin V–positive cells are significantly decreased in the p53 knockout compared with the wild type. **, P < 0.01. (right) Deletion of p53 was confirmed by Western blotting with anti-p53 using a lung protein lysate. Molecular masses are given in kilodaltons. Data are shown as means ± SEM. WT, wild type.

Article Snippet: Specificity of anti-p53 staining was tested by staining with anti-p53 preabsorbed with recombinant p53 (Santa Cruz Biotechnology, Inc.) as shown in Fig. S4.

Techniques: Expressing, Staining, Marker, Fluorescence, Knock-Out, Western Blot