Structured Review

Santa Cruz Biotechnology goat polyclonal anti p2y 2 r
Rabbit <t>P2Y</t> 2 -R cDNA. A : Nucleotide sequence and deduced amino acid sequence of cloned rabbit P2Y 2 -R are shown. siRNA-target sequences are indicated with solid lines. B : Alignment of the rabbit deduced amino acid sequence with the human P2Y 2 -R sequence is displayed. Identical amino acids are shaded. Protein database searches revealed that the deduced sequence had the highest amino acid identity with human P2Y 2 -R (93%). These sequences are available under GenBank accession number EU886321 (rabbit) and NP_788086.1 (human).
Goat Polyclonal Anti P2y 2 R, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Silencing of P2Y 2 receptor delays Ap 4 A - corneal re-epithelialization process"

Article Title: Silencing of P2Y 2 receptor delays Ap 4 A - corneal re-epithelialization process

Journal: Molecular Vision

doi:

Rabbit P2Y 2 -R cDNA. A : Nucleotide sequence and deduced amino acid sequence of cloned rabbit P2Y 2 -R are shown. siRNA-target sequences are indicated with solid lines. B : Alignment of the rabbit deduced amino acid sequence with the human P2Y 2 -R sequence is displayed. Identical amino acids are shaded. Protein database searches revealed that the deduced sequence had the highest amino acid identity with human P2Y 2 -R (93%). These sequences are available under GenBank accession number EU886321 (rabbit) and NP_788086.1 (human).
Figure Legend Snippet: Rabbit P2Y 2 -R cDNA. A : Nucleotide sequence and deduced amino acid sequence of cloned rabbit P2Y 2 -R are shown. siRNA-target sequences are indicated with solid lines. B : Alignment of the rabbit deduced amino acid sequence with the human P2Y 2 -R sequence is displayed. Identical amino acids are shaded. Protein database searches revealed that the deduced sequence had the highest amino acid identity with human P2Y 2 -R (93%). These sequences are available under GenBank accession number EU886321 (rabbit) and NP_788086.1 (human).

Techniques Used: Sequencing, Clone Assay

P2Y 2 -R immunostaining of transfected cells and treated corneas. A : SIRC cells were incubated for 72 h with transfection reagent alone (control) or with P2Y 2 -R siRNA #2 and then processed for ICC (40X magnification). B : The chart shows P2Y 2 -R staining intensity quantification of control and P2Y 2 -R siRNA transfected cells 72 h post-transfection. C : A series of micrographs shows the P2Y 2 -R signal in corneas treated with 0.9% saline, 100 μM Ap 4 A, and siRNA+100 μM Ap 4 A while we can observed the nuclear staining for DAPI in blue (40X). D : The graph shows the P2Y 2 -R intensity signal in the three different treatments at 24 h and 36 h after wounding. Three asterisks mean p<0.001 when compared to the control. Green fluorescence (FITC) localizes P2Y 2 -R.
Figure Legend Snippet: P2Y 2 -R immunostaining of transfected cells and treated corneas. A : SIRC cells were incubated for 72 h with transfection reagent alone (control) or with P2Y 2 -R siRNA #2 and then processed for ICC (40X magnification). B : The chart shows P2Y 2 -R staining intensity quantification of control and P2Y 2 -R siRNA transfected cells 72 h post-transfection. C : A series of micrographs shows the P2Y 2 -R signal in corneas treated with 0.9% saline, 100 μM Ap 4 A, and siRNA+100 μM Ap 4 A while we can observed the nuclear staining for DAPI in blue (40X). D : The graph shows the P2Y 2 -R intensity signal in the three different treatments at 24 h and 36 h after wounding. Three asterisks mean p<0.001 when compared to the control. Green fluorescence (FITC) localizes P2Y 2 -R.

Techniques Used: Immunostaining, Transfection, Incubation, Staining, Fluorescence

Quantification of P2Y 2 -R mRNA levels by qRT–PCR analysis. Corneal impression cytology samples were collected at 48, 72, and 96 h after the first siRNA instillation and then submitted to total RNA isolation. After retrotranscription, we measured P2Y 2 -R mRNA knockdown in corneas treated with siRNA #2. Values derived from qRT–PCR results from untreated corneas are set as 100%, and the relative expressions of corneas treated with siRNA are indicated. Three asterisks indicate p<0.001 when compared to the control.
Figure Legend Snippet: Quantification of P2Y 2 -R mRNA levels by qRT–PCR analysis. Corneal impression cytology samples were collected at 48, 72, and 96 h after the first siRNA instillation and then submitted to total RNA isolation. After retrotranscription, we measured P2Y 2 -R mRNA knockdown in corneas treated with siRNA #2. Values derived from qRT–PCR results from untreated corneas are set as 100%, and the relative expressions of corneas treated with siRNA are indicated. Three asterisks indicate p<0.001 when compared to the control.

Techniques Used: Quantitative RT-PCR, Isolation, Derivative Assay

Effect of P2Y 2 -R siRNA on Ap 4 A-induced migration of corneal epithelial cells. SIRC cells were incubated for 72 h with transfection reagent alone (control), P2Y 2 -R siRNA #1, or P2Y 2 -R siRNA #2 and then wounded with a pipette tip in the presence or absence of Ap 4 A. The graphs ( A and B ) show the variation of the wounded area versus time and the estimated migration rates (EMR) of cells transfected with siRNA 1 and siRNA 2, respectively, in the presence of Ap 4 A ( P2Y 2 -R siRNA+Ap 4 A). Ap 4 A accelerated the rate of healing in cells transfected with transfection reagent alone (control+Ap 4 A) compared with the rate of both P2Y 2 -R siRNA-transfected cells in the presence of Ap 4 A ( P2Y 2 -R siRNA+Ap 4 A). Three asterisks indicate p<0.0001 when compared to the control, and two asterisks indicate p<0.001 when compared to Ap 4 A alone.
Figure Legend Snippet: Effect of P2Y 2 -R siRNA on Ap 4 A-induced migration of corneal epithelial cells. SIRC cells were incubated for 72 h with transfection reagent alone (control), P2Y 2 -R siRNA #1, or P2Y 2 -R siRNA #2 and then wounded with a pipette tip in the presence or absence of Ap 4 A. The graphs ( A and B ) show the variation of the wounded area versus time and the estimated migration rates (EMR) of cells transfected with siRNA 1 and siRNA 2, respectively, in the presence of Ap 4 A ( P2Y 2 -R siRNA+Ap 4 A). Ap 4 A accelerated the rate of healing in cells transfected with transfection reagent alone (control+Ap 4 A) compared with the rate of both P2Y 2 -R siRNA-transfected cells in the presence of Ap 4 A ( P2Y 2 -R siRNA+Ap 4 A). Three asterisks indicate p<0.0001 when compared to the control, and two asterisks indicate p<0.001 when compared to Ap 4 A alone.

Techniques Used: Migration, Incubation, Transfection, Transferring

Effect of P2Y 2 -R siRNA on Ap 4 A-mediated corneal wound healing. A : A representative sequence of the progress in the corneal wound in the three different treatments (0.9% saline, 100 μM Ap 4 A, and siRNA+100 μM Ap 4 A) is shown. B : The graphs show the variation of the wounded area versus time and the estimated migration rates (EMR) in the three treatments. An asterisk means p<0.05 when compared to the control, and a double asterisk indicates p<0.01 when compared to the control.
Figure Legend Snippet: Effect of P2Y 2 -R siRNA on Ap 4 A-mediated corneal wound healing. A : A representative sequence of the progress in the corneal wound in the three different treatments (0.9% saline, 100 μM Ap 4 A, and siRNA+100 μM Ap 4 A) is shown. B : The graphs show the variation of the wounded area versus time and the estimated migration rates (EMR) in the three treatments. An asterisk means p<0.05 when compared to the control, and a double asterisk indicates p<0.01 when compared to the control.

Techniques Used: Sequencing, Migration


Structured Review

Santa Cruz Biotechnology p2y 2 r shrna plasmid
Caspase-1 activity or IL-1β secretion induced by TNF-α or ATP is significantly suppressed by <t>P2Y</t> <t>2</t> <t>R</t> knockdown or apyrase (an enzyme that rapidly hydrolyzes extracellular nucleotides) in MDA-MB-231 or RT-R-MDA-MB-231 cells. (A) P2Y 2 R mRNA levels were analyzed by RT-PCR to confirm the efficiency of the knockdown in control siRNA (siCTRL)- or P2Y 2 R siRNA (siP2Y 2 R)-transfected cells. (B and C) siCTRL or siP2Y 2 R-transfected cells were stimulated with TNF-α (10 ng/ml) or ATP (10 µ M). (B) Caspase-1 activity and (C) IL-1β secretion were measured 3 h and 24 h after treatment, respectively, as described in the Materials and methods. (D and E) The cells were pre-treated with 10 U/ml apyrase for 1 h and stimulated with TNF-α (10 ng/ml). (D) Caspase-1 activity and (E) IL-1β secretion were measured as described in (A). The values represent the means ± SEM of 3 independent experiments. ** P<0.01, compared to the control (CTRL) of each cells; # P<0.05 and ## P<0.05, compared to the TNF-α treatment of each of the cells; † P<0.05 and ‡ P<0.01, compared to the ATP treatment of each of the cells; § P<0.05 and §§ P<0.01, significance between the MDA-MB-231 and RT-R-MDA-MB-231 cells. ATP, adenosine triphosphate; RT-R, radiotherapy-resistant; IL-1β, interleukin-1β; TNF-α, tumor necrosis factor-α.
P2y 2 R Shrna Plasmid, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Price from $9.99 to $1999.99
p2y 2 r shrna plasmid - by Bioz Stars, 2023-02
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1) Product Images from "P2Y 2 R-mediated inflammasome activation is involved in tumor progression in breast cancer cells and in radiotherapy-resistant breast cancer"

Article Title: P2Y 2 R-mediated inflammasome activation is involved in tumor progression in breast cancer cells and in radiotherapy-resistant breast cancer

Journal: International Journal of Oncology

doi: 10.3892/ijo.2018.4552

Caspase-1 activity or IL-1β secretion induced by TNF-α or ATP is significantly suppressed by P2Y 2 R knockdown or apyrase (an enzyme that rapidly hydrolyzes extracellular nucleotides) in MDA-MB-231 or RT-R-MDA-MB-231 cells. (A) P2Y 2 R mRNA levels were analyzed by RT-PCR to confirm the efficiency of the knockdown in control siRNA (siCTRL)- or P2Y 2 R siRNA (siP2Y 2 R)-transfected cells. (B and C) siCTRL or siP2Y 2 R-transfected cells were stimulated with TNF-α (10 ng/ml) or ATP (10 µ M). (B) Caspase-1 activity and (C) IL-1β secretion were measured 3 h and 24 h after treatment, respectively, as described in the Materials and methods. (D and E) The cells were pre-treated with 10 U/ml apyrase for 1 h and stimulated with TNF-α (10 ng/ml). (D) Caspase-1 activity and (E) IL-1β secretion were measured as described in (A). The values represent the means ± SEM of 3 independent experiments. ** P<0.01, compared to the control (CTRL) of each cells; # P<0.05 and ## P<0.05, compared to the TNF-α treatment of each of the cells; † P<0.05 and ‡ P<0.01, compared to the ATP treatment of each of the cells; § P<0.05 and §§ P<0.01, significance between the MDA-MB-231 and RT-R-MDA-MB-231 cells. ATP, adenosine triphosphate; RT-R, radiotherapy-resistant; IL-1β, interleukin-1β; TNF-α, tumor necrosis factor-α.
Figure Legend Snippet: Caspase-1 activity or IL-1β secretion induced by TNF-α or ATP is significantly suppressed by P2Y 2 R knockdown or apyrase (an enzyme that rapidly hydrolyzes extracellular nucleotides) in MDA-MB-231 or RT-R-MDA-MB-231 cells. (A) P2Y 2 R mRNA levels were analyzed by RT-PCR to confirm the efficiency of the knockdown in control siRNA (siCTRL)- or P2Y 2 R siRNA (siP2Y 2 R)-transfected cells. (B and C) siCTRL or siP2Y 2 R-transfected cells were stimulated with TNF-α (10 ng/ml) or ATP (10 µ M). (B) Caspase-1 activity and (C) IL-1β secretion were measured 3 h and 24 h after treatment, respectively, as described in the Materials and methods. (D and E) The cells were pre-treated with 10 U/ml apyrase for 1 h and stimulated with TNF-α (10 ng/ml). (D) Caspase-1 activity and (E) IL-1β secretion were measured as described in (A). The values represent the means ± SEM of 3 independent experiments. ** P<0.01, compared to the control (CTRL) of each cells; # P<0.05 and ## P<0.05, compared to the TNF-α treatment of each of the cells; † P<0.05 and ‡ P<0.01, compared to the ATP treatment of each of the cells; § P<0.05 and §§ P<0.01, significance between the MDA-MB-231 and RT-R-MDA-MB-231 cells. ATP, adenosine triphosphate; RT-R, radiotherapy-resistant; IL-1β, interleukin-1β; TNF-α, tumor necrosis factor-α.

Techniques Used: Activity Assay, Reverse Transcription Polymerase Chain Reaction, Transfection

Comparisons of ATP release, P2Y 2 R activity and invasiveness between breast cancer cells and RT-R breast cancer cells. (A) ATP released into the extracellular medium was measured using the ENLITEN ATP assay system kit, as described in the Materials and methods. The values represent the means ± SEM of 3 independent experiments (H, HEPES buffer only). ** P<0.01, compared to the control (CTRL) of each parent breast cancer cell; ## P<0.01, compared to the CTRL of each RT-R breast cancer cells. (B) [Ca 2+ ] i levels were determined in breast cancer cells and RT-R breast cancer cells to measure P2Y 2 R activities. Arrows indicate the points at which ATP (10 µ M) was added. The values represent the means ± SEM from 3 independent determinations. *P<0.05 and ** P<0.01, compared to the CTRL of MDA-MB-231 cells. (C) RT-R-breast cancer cells were treated with ATP for 6 h, and Matrigel invasion assay was performed as described in the Materials and methods. The values represent the means ± SEM of 3 independent experiments. ** P<0.01, compared to the CTRL of RT-R-MDA-MB-231 cells; ## P<0.01, compared to ATP-treated RT-R-MDA-MB-231 cells. Scale bar, 50 µ m. ATP, adenosine triphosphate; RT-R, radiotherapy-resistant.
Figure Legend Snippet: Comparisons of ATP release, P2Y 2 R activity and invasiveness between breast cancer cells and RT-R breast cancer cells. (A) ATP released into the extracellular medium was measured using the ENLITEN ATP assay system kit, as described in the Materials and methods. The values represent the means ± SEM of 3 independent experiments (H, HEPES buffer only). ** P<0.01, compared to the control (CTRL) of each parent breast cancer cell; ## P<0.01, compared to the CTRL of each RT-R breast cancer cells. (B) [Ca 2+ ] i levels were determined in breast cancer cells and RT-R breast cancer cells to measure P2Y 2 R activities. Arrows indicate the points at which ATP (10 µ M) was added. The values represent the means ± SEM from 3 independent determinations. *P<0.05 and ** P<0.01, compared to the CTRL of MDA-MB-231 cells. (C) RT-R-breast cancer cells were treated with ATP for 6 h, and Matrigel invasion assay was performed as described in the Materials and methods. The values represent the means ± SEM of 3 independent experiments. ** P<0.01, compared to the CTRL of RT-R-MDA-MB-231 cells; ## P<0.01, compared to ATP-treated RT-R-MDA-MB-231 cells. Scale bar, 50 µ m. ATP, adenosine triphosphate; RT-R, radiotherapy-resistant.

Techniques Used: Activity Assay, ATP Assay, Invasion Assay

MMP-9 activity is modulated by caspase-1 in a P2Y 2 R-dependent manner, in MDA-MB-231 or RT-R-MDA-MB-231 cells. (A) Cells were pre-treated with Ac-YVAD-CMK and then stimulated with TNF-α or ATP for 6 h. MMP-9 gelatinase activity was determined as described in the Materials and methods and quantified. The values represent the means ± SEM of 3 independent experiments. * P<0.05 and ** P<0.01, compared to the control (CTRL) of each of the cells; ## P<0.05, compared to the TNF-α treatment of each of the cells; ‡ P<0.05, compared to the ATP treatment of each of the cells; § P<0.05, comparison between the MDA-MB-231 and RT-R-MDA-MB-231 cells. (B) siCTRL- or siP2Y 2 R-transfected RT-R-MDA-MB-231 cells were pre-treated with AR-C 118925XX (AR), a specific P2Y 2 R antagonist or not. The cells were then stimulated with ATP, and MMP-9 gelatinase activity was determined as described in the Materials and methods. The values represent the means ± SEM of 3 independent experiments. ** P<0.05, compared to the CTRL; ## P<0.01, compared to ATP treatment. ATP, adenosine triphosphate; RT-R, radiotherapy-resistant; IL-1β, interleukin-1β; TNF-α, tumor necrosis factor-α; MMP-9, matrix metalloproteinase-9.
Figure Legend Snippet: MMP-9 activity is modulated by caspase-1 in a P2Y 2 R-dependent manner, in MDA-MB-231 or RT-R-MDA-MB-231 cells. (A) Cells were pre-treated with Ac-YVAD-CMK and then stimulated with TNF-α or ATP for 6 h. MMP-9 gelatinase activity was determined as described in the Materials and methods and quantified. The values represent the means ± SEM of 3 independent experiments. * P<0.05 and ** P<0.01, compared to the control (CTRL) of each of the cells; ## P<0.05, compared to the TNF-α treatment of each of the cells; ‡ P<0.05, compared to the ATP treatment of each of the cells; § P<0.05, comparison between the MDA-MB-231 and RT-R-MDA-MB-231 cells. (B) siCTRL- or siP2Y 2 R-transfected RT-R-MDA-MB-231 cells were pre-treated with AR-C 118925XX (AR), a specific P2Y 2 R antagonist or not. The cells were then stimulated with ATP, and MMP-9 gelatinase activity was determined as described in the Materials and methods. The values represent the means ± SEM of 3 independent experiments. ** P<0.05, compared to the CTRL; ## P<0.01, compared to ATP treatment. ATP, adenosine triphosphate; RT-R, radiotherapy-resistant; IL-1β, interleukin-1β; TNF-α, tumor necrosis factor-α; MMP-9, matrix metalloproteinase-9.

Techniques Used: Activity Assay, Transfection

Suppression of P2Y 2 R reduced RT-R-breast cancer cell growth by regulating MMP-9 expression in an in vivo mouse model. Athymic nude mice were divided into 2 groups and injected subcutaneously with empty vector-transfected RT-R-MDA-MB-231 cells (RT-R-MDA-MB-231-EV; n=5) or P2Y 2 R-shRNA- transfected RT-R-MDA-MB-231 cells (RT-R-MDA-MB-231-P2Y 2 R-shRNA; n=5) (5×10 6 cells/100 µ l of serum-free medium). RT-R-MDA-MB-231-EV-injected or RT-R-MDA-MB-231-P2Y 2 R shRNA-injected animals were sacrificed on day 61. (A) Tumor volumes and (B) body weights were measured every 3 days during tumor development, and the bar graphs were presented at the end of 61st day. (C) Tumor tissue sections were stained with anti-MMP-9 antibody (scale bar, 100 µ m), and the sections were counterstained with Mayer’s hematoxylin solution. (D) IL-1β levels in the serum were analyzed as described in the Materials and methods (n=3). (E) Schematic representation of the proposed role of P2Y 2 R in RT-R-breast cancer cell progression and invasiveness through interaction with the inflammasome.RT-R, radiotherapy-resistant; P2Y 2 R, P2Y purinergic receptor 2.
Figure Legend Snippet: Suppression of P2Y 2 R reduced RT-R-breast cancer cell growth by regulating MMP-9 expression in an in vivo mouse model. Athymic nude mice were divided into 2 groups and injected subcutaneously with empty vector-transfected RT-R-MDA-MB-231 cells (RT-R-MDA-MB-231-EV; n=5) or P2Y 2 R-shRNA- transfected RT-R-MDA-MB-231 cells (RT-R-MDA-MB-231-P2Y 2 R-shRNA; n=5) (5×10 6 cells/100 µ l of serum-free medium). RT-R-MDA-MB-231-EV-injected or RT-R-MDA-MB-231-P2Y 2 R shRNA-injected animals were sacrificed on day 61. (A) Tumor volumes and (B) body weights were measured every 3 days during tumor development, and the bar graphs were presented at the end of 61st day. (C) Tumor tissue sections were stained with anti-MMP-9 antibody (scale bar, 100 µ m), and the sections were counterstained with Mayer’s hematoxylin solution. (D) IL-1β levels in the serum were analyzed as described in the Materials and methods (n=3). (E) Schematic representation of the proposed role of P2Y 2 R in RT-R-breast cancer cell progression and invasiveness through interaction with the inflammasome.RT-R, radiotherapy-resistant; P2Y 2 R, P2Y purinergic receptor 2.

Techniques Used: Expressing, In Vivo, Injection, Plasmid Preparation, Transfection, shRNA, Staining


Structured Review

Santa Cruz Biotechnology p2y 2 r
Summary of <t> P2Y 2 R </t> study HCPS decedent population.
P2y 2 R, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 86 stars, based on 1 article reviews
Price from $9.99 to $1999.99
p2y 2 r - by Bioz Stars, 2023-02
86/100 stars

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1) Product Images from "Upregulation of P2Y 2 R, Active uPA, and PAI-1 Are Essential Components of Hantavirus Cardiopulmonary Syndrome"

Article Title: Upregulation of P2Y 2 R, Active uPA, and PAI-1 Are Essential Components of Hantavirus Cardiopulmonary Syndrome

Journal: Frontiers in Cellular and Infection Microbiology

doi: 10.3389/fcimb.2018.00169

Summary of  P2Y 2 R  study HCPS decedent population.
Figure Legend Snippet: Summary of P2Y 2 R study HCPS decedent population.

Techniques Used:

P2Y 2 R expression is significantly upregulated in HCPS compared to controls. RNA was extracted from lung tissues samples with the Qiagen RNeasy FFPE kit (cat# 73504) and quantified with a Thermo Fisher Scientific Nanodrop. P2Y 2 R expression levels were measured by the TaqMan assay using a WT P2Y 2 R plasmid as standard. P2Y 2 R copy #s were normalized to total RNA extracted from the embedded tissues. Each sample slice was measured in triplicate. (A) The plot of P2Y 2 R for each HCPS patient, shown on the x-axis as H1-H22. Each data point per patient represents a triplicate measurement of P2Y 2 R expression in a 10 μm slice cut from the FFPE blocks. The number of segments analyzed is shown for each case above the replicate data points (e.g., n = 11 for H1). Ninety-seven distinct segments from all cases were analyzed. Lung tissues from two decedents (H4, and H9) were not available. The error bars represent the median and range. For all HCPS samples, the minimum, median, maximum and mean values were 0.0055, 2.10, 24.88, and 4.1 ± 0.48 (SEM). (B,C) Plot of P2Y 2 R for pneumonia (P) and gunshot wound cases (GSW). The experimental conditions are similar to those described for HCPS. Cases for which no lung tissue samples were available were excluded from the graph. For all pneumonia tissue segment samples ( n = 39), the minimum, median, maximum and mean values were 0.0005, 0.07, 4.4, 0.54 ± 0.16. For all GSW samples ( n = 21), the minimum, median, maximum and mean values were 0.0052, 0.18, 2.4, and 0.46 ± 0.10 (SEM).
Figure Legend Snippet: P2Y 2 R expression is significantly upregulated in HCPS compared to controls. RNA was extracted from lung tissues samples with the Qiagen RNeasy FFPE kit (cat# 73504) and quantified with a Thermo Fisher Scientific Nanodrop. P2Y 2 R expression levels were measured by the TaqMan assay using a WT P2Y 2 R plasmid as standard. P2Y 2 R copy #s were normalized to total RNA extracted from the embedded tissues. Each sample slice was measured in triplicate. (A) The plot of P2Y 2 R for each HCPS patient, shown on the x-axis as H1-H22. Each data point per patient represents a triplicate measurement of P2Y 2 R expression in a 10 μm slice cut from the FFPE blocks. The number of segments analyzed is shown for each case above the replicate data points (e.g., n = 11 for H1). Ninety-seven distinct segments from all cases were analyzed. Lung tissues from two decedents (H4, and H9) were not available. The error bars represent the median and range. For all HCPS samples, the minimum, median, maximum and mean values were 0.0055, 2.10, 24.88, and 4.1 ± 0.48 (SEM). (B,C) Plot of P2Y 2 R for pneumonia (P) and gunshot wound cases (GSW). The experimental conditions are similar to those described for HCPS. Cases for which no lung tissue samples were available were excluded from the graph. For all pneumonia tissue segment samples ( n = 39), the minimum, median, maximum and mean values were 0.0005, 0.07, 4.4, 0.54 ± 0.16. For all GSW samples ( n = 21), the minimum, median, maximum and mean values were 0.0052, 0.18, 2.4, and 0.46 ± 0.10 (SEM).

Techniques Used: Expressing, TaqMan Assay, Plasmid Preparation

P2Y 2 R expression is significantly upregulated in HCPS compared to controls. Plots of (A) Mean and (B) Natural log-transformed values of P2Y 2 R copy numbers in 10 μm slices cut from the FFPE lung tissue blocks versus body mass index or BMI of Gunshot, pneumonia and HCPS cases. Error bars represent measurements of P2Y 2 R in slices from different segments of lung tissue from each case (indicated in Figure ). Statistical analysis was performed on natural log-transformed data to approximate a normal distribution. See text for details.
Figure Legend Snippet: P2Y 2 R expression is significantly upregulated in HCPS compared to controls. Plots of (A) Mean and (B) Natural log-transformed values of P2Y 2 R copy numbers in 10 μm slices cut from the FFPE lung tissue blocks versus body mass index or BMI of Gunshot, pneumonia and HCPS cases. Error bars represent measurements of P2Y 2 R in slices from different segments of lung tissue from each case (indicated in Figure ). Statistical analysis was performed on natural log-transformed data to approximate a normal distribution. See text for details.

Techniques Used: Expressing, Transformation Assay

Histological analysis of lung samples of autopsied HPCS cases. Shown are representative sections of lungs stained with Hematoxylin and Eosin (H&E). (A) Sample of lung parenchyma of a gunshot wound decedent, showing open alveolar space (case GSW1). (B) Lung sample of an HCPS patient (case H21 Block A2) highlighting hyaline alveolar membranes (arrows), infiltrates of various immune cells immunoblasts, macrophages and neutrophils (few) (original magnification × 400). (C) Lung sample (case H1 Block D), highlighting focal deposits of macrophages (circles) and intraalveolar extravasated red blood cells (asterisk). (D) Lung sample (H1 case Block I) highlighting extensive congestion and intraalveolar edema with mild interstitial inflammation compared to (case H1 Block D) shown in (C) . The values of 5.5 and 0.4 in (C , D) respectively refer to P2Y 2 R RNA measured in the TaqMan assay (original magnifications for C,D × 200).
Figure Legend Snippet: Histological analysis of lung samples of autopsied HPCS cases. Shown are representative sections of lungs stained with Hematoxylin and Eosin (H&E). (A) Sample of lung parenchyma of a gunshot wound decedent, showing open alveolar space (case GSW1). (B) Lung sample of an HCPS patient (case H21 Block A2) highlighting hyaline alveolar membranes (arrows), infiltrates of various immune cells immunoblasts, macrophages and neutrophils (few) (original magnification × 400). (C) Lung sample (case H1 Block D), highlighting focal deposits of macrophages (circles) and intraalveolar extravasated red blood cells (asterisk). (D) Lung sample (H1 case Block I) highlighting extensive congestion and intraalveolar edema with mild interstitial inflammation compared to (case H1 Block D) shown in (C) . The values of 5.5 and 0.4 in (C , D) respectively refer to P2Y 2 R RNA measured in the TaqMan assay (original magnifications for C,D × 200).

Techniques Used: Staining, Blocking Assay, TaqMan Assay

Comparison of serial sections from two different lung segments expressing low (s1) and high levels of P2Y 2 R (s2) from HCPS patient (case H7 Block C). (A) Weak staining for P2Y 2 R segment 1 corresponds to low P2Y 2 R copy numbers (0.1 TaqMan). (B) Relatively high P2Y 2 R copy numbers (9.3 TaqMan) is associated with a robust positive staining for P2Y 2 R in macrophages and endothelial cell-proximal type II pneumocytes. (C) Weak staining for SNV in segment 1. (D) Stronger positive staining for SNV in type II-pneumocytes and endothelial cells. (E,F) Comparative staining for uPA staining in s1 and s2 segments stronger staining in s2 associated with higher macrophage deposits (circle). (G,H) Relative staining for neutrophil elastase in s1 and s2 indicates stronger staining for neutrophils related to higher P2Y 2 R (original magnification x200).
Figure Legend Snippet: Comparison of serial sections from two different lung segments expressing low (s1) and high levels of P2Y 2 R (s2) from HCPS patient (case H7 Block C). (A) Weak staining for P2Y 2 R segment 1 corresponds to low P2Y 2 R copy numbers (0.1 TaqMan). (B) Relatively high P2Y 2 R copy numbers (9.3 TaqMan) is associated with a robust positive staining for P2Y 2 R in macrophages and endothelial cell-proximal type II pneumocytes. (C) Weak staining for SNV in segment 1. (D) Stronger positive staining for SNV in type II-pneumocytes and endothelial cells. (E,F) Comparative staining for uPA staining in s1 and s2 segments stronger staining in s2 associated with higher macrophage deposits (circle). (G,H) Relative staining for neutrophil elastase in s1 and s2 indicates stronger staining for neutrophils related to higher P2Y 2 R (original magnification x200).

Techniques Used: Expressing, Blocking Assay, Staining

P2Y 2 R, uPA, PAI-1, and TF colocalize with macrophages in serial HCPS lung tissue sections. (A) Micrograph of lung tissue of HCPS patient (case H7 Block A) stained with hematoxylin and eosin (H&E) showing alveolar macrophages, neutrophils, and lymphocytes. (B) Immunohistochemical-detection of P2Y 2 R expression on macrophages and type 2 pneumocytes (H70 antibody: SC-20124). (C) Positive antigen staining for uPA in type II pneumocytes, hyaline membranes, alveolar macrophage (circles) (original magnification × 400). (D) H & E image of lung section of an HCPS patient (case H1 Block D) highlighting hyaline alveolar membranes, and macrophages in circles. (E,F) Serial Lung sections of case H1 Block D reveal cellular localization of PAI-1 and TF. PAI-1 mostly associated with macrophages and type II pneumocytes. PAI-1 was detected with the H135 antibody, Sc-8979 (Santa Cruz). TF staining was localized with alveolar macrophages or distributed in type II pneumocytes. Tissue factor (TF) was detected by a rabbit monoclonal antibody EPR8986 ab151748 (Abcam) (original magnification for all slides × 400).
Figure Legend Snippet: P2Y 2 R, uPA, PAI-1, and TF colocalize with macrophages in serial HCPS lung tissue sections. (A) Micrograph of lung tissue of HCPS patient (case H7 Block A) stained with hematoxylin and eosin (H&E) showing alveolar macrophages, neutrophils, and lymphocytes. (B) Immunohistochemical-detection of P2Y 2 R expression on macrophages and type 2 pneumocytes (H70 antibody: SC-20124). (C) Positive antigen staining for uPA in type II pneumocytes, hyaline membranes, alveolar macrophage (circles) (original magnification × 400). (D) H & E image of lung section of an HCPS patient (case H1 Block D) highlighting hyaline alveolar membranes, and macrophages in circles. (E,F) Serial Lung sections of case H1 Block D reveal cellular localization of PAI-1 and TF. PAI-1 mostly associated with macrophages and type II pneumocytes. PAI-1 was detected with the H135 antibody, Sc-8979 (Santa Cruz). TF staining was localized with alveolar macrophages or distributed in type II pneumocytes. Tissue factor (TF) was detected by a rabbit monoclonal antibody EPR8986 ab151748 (Abcam) (original magnification for all slides × 400).

Techniques Used: Blocking Assay, Staining, Immunohistochemical staining, Expressing

Immunohistologic examination of lobar pneumonia patient (case P6 Block B) shows strong staining for neutrophil elastase, but poor staining for P2Y 2 R and uPA. (A) Micrograph of lung tissue stained with hematoxylin and eosin (H&E) showing numerous neutrophils congest alveolar space and relatively few macrophages and lymphocytes. (B) Intense staining for polymorphonuclear (PMN) leukocytes with antibodies against neutrophil elastase (Abcam ab68672). (C) Weak staining for P2Y 2 R expression (H70 antibody: SC-20124), deficiency of P2Y 2 R is agreement with low P2Y 2 R (TaqMan measurement). (D) Weak staining for uPA is correlated to low macrophage expression.
Figure Legend Snippet: Immunohistologic examination of lobar pneumonia patient (case P6 Block B) shows strong staining for neutrophil elastase, but poor staining for P2Y 2 R and uPA. (A) Micrograph of lung tissue stained with hematoxylin and eosin (H&E) showing numerous neutrophils congest alveolar space and relatively few macrophages and lymphocytes. (B) Intense staining for polymorphonuclear (PMN) leukocytes with antibodies against neutrophil elastase (Abcam ab68672). (C) Weak staining for P2Y 2 R expression (H70 antibody: SC-20124), deficiency of P2Y 2 R is agreement with low P2Y 2 R (TaqMan measurement). (D) Weak staining for uPA is correlated to low macrophage expression.

Techniques Used: Blocking Assay, Staining, Expressing

Model of the development of P2Y 2 R, scuPA, and PAI activity during HCPS (alveolus image was adapted from Kumar et al., ). Macrophages and dendritic cells recognize hantavirus after inhalation. Extracellular nucleotides increase under inflammatory conditions and activate P2 receptor family subtypes, including P2X 7 and P2Y 2 R (Hechler and Gachet, ). Activation of P2X 7 stimulates the release of IL-1β and upregulates P2Y 2 R in myeloid and endothelial cells. P2Y 2 R promotes, SNV infectivity, monocyte adherence to endothelial cells through VCAM-1 and contributes to chemotaxis. TNFα primarily by myeloid lineage cells and IFN-γ released by lymphocytes during HCPS (Mori et al., ; Safronetz et al., ) have been shown to upregulate soluble uPAR and expression of membrane-bound uPAR. Thus increased expression of uPAR and uPA is expected to enhance the proteolytic activity of chemotactic cells during HCPS. PAI-1 binds poorly to scuPA-uPAR complexes and might induce appositive feedback loop for increased protease activity.
Figure Legend Snippet: Model of the development of P2Y 2 R, scuPA, and PAI activity during HCPS (alveolus image was adapted from Kumar et al., ). Macrophages and dendritic cells recognize hantavirus after inhalation. Extracellular nucleotides increase under inflammatory conditions and activate P2 receptor family subtypes, including P2X 7 and P2Y 2 R (Hechler and Gachet, ). Activation of P2X 7 stimulates the release of IL-1β and upregulates P2Y 2 R in myeloid and endothelial cells. P2Y 2 R promotes, SNV infectivity, monocyte adherence to endothelial cells through VCAM-1 and contributes to chemotaxis. TNFα primarily by myeloid lineage cells and IFN-γ released by lymphocytes during HCPS (Mori et al., ; Safronetz et al., ) have been shown to upregulate soluble uPAR and expression of membrane-bound uPAR. Thus increased expression of uPAR and uPA is expected to enhance the proteolytic activity of chemotactic cells during HCPS. PAI-1 binds poorly to scuPA-uPAR complexes and might induce appositive feedback loop for increased protease activity.

Techniques Used: Activity Assay, Activation Assay, Infection, Chemotaxis Assay, Expressing


Structured Review

Santa Cruz Biotechnology p2y 2 r
Switchblade model for integrin activation (adapted from ) and schematic presenting the hypothesis that cis interaction of α IIb β 3 integrin and RGD <t>P2Y</t> <t>2</t> <t>R</t> mediates integrin activation initiated by binding of SNV to the PSI domain. (A) 1) Structure of an inactive integrin. 2–3) Intracellular signaling (inside-out) induces integrin activation mediated by binding of adaptor proteins (such as talin) to the extended conformation with open head-piece bound to soluble and immobilized ligands (see the text for details). 4) Development of mechanochemical force selectively transduced through the β subunit. Integrin binding to immobilized ligand resists lateral translation and causes an increase in force (indicated by arrows) and promotes separation of the α− and β-subunit transmembrane domains. (B) 5) P2Y 2 R interacts in cis with α IIb β 3 integrin. 6) SNV occupancy of the PSI domain induces an increase in integrin affinity for cis - RGD P2Y 2 R (based on AFM measurements). Integrin extension exerts a membrane normal/lateral pulling force, due to height differences between the membrane proximal RGD P2Y 2 R, and the unbending integrin ( Takagi et al. , 2002 ; Chigaev et al. , 2003, 2015); Outside-in signaling. 7) Recruitment of talin and other adhesion molecules increases force transduction, which is terminated by rupture of RGD interaction. Full extension of α IIb β 3 integrin causes rupture of cis interaction, indicated by PAC1 staining of cells. 8) Cessation of tensile force, and loss of intracellular link to actin, leads to exchange of adhesion proteins, which are replaced by adaptor proteins (e.g., clathrin and Dab2) for integrin endocytosis ( Yu et al. , 2015 ).
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1) Product Images from "Low-affinity binding in cis to P2Y 2 R mediates force-dependent integrin activation during hantavirus infection"

Article Title: Low-affinity binding in cis to P2Y 2 R mediates force-dependent integrin activation during hantavirus infection

Journal: Molecular Biology of the Cell

doi: 10.1091/mbc.E17-01-0082

Switchblade model for integrin activation (adapted from ) and schematic presenting the hypothesis that cis interaction of α IIb β 3 integrin and RGD P2Y 2 R mediates integrin activation initiated by binding of SNV to the PSI domain. (A) 1) Structure of an inactive integrin. 2–3) Intracellular signaling (inside-out) induces integrin activation mediated by binding of adaptor proteins (such as talin) to the extended conformation with open head-piece bound to soluble and immobilized ligands (see the text for details). 4) Development of mechanochemical force selectively transduced through the β subunit. Integrin binding to immobilized ligand resists lateral translation and causes an increase in force (indicated by arrows) and promotes separation of the α− and β-subunit transmembrane domains. (B) 5) P2Y 2 R interacts in cis with α IIb β 3 integrin. 6) SNV occupancy of the PSI domain induces an increase in integrin affinity for cis - RGD P2Y 2 R (based on AFM measurements). Integrin extension exerts a membrane normal/lateral pulling force, due to height differences between the membrane proximal RGD P2Y 2 R, and the unbending integrin ( Takagi et al. , 2002 ; Chigaev et al. , 2003, 2015); Outside-in signaling. 7) Recruitment of talin and other adhesion molecules increases force transduction, which is terminated by rupture of RGD interaction. Full extension of α IIb β 3 integrin causes rupture of cis interaction, indicated by PAC1 staining of cells. 8) Cessation of tensile force, and loss of intracellular link to actin, leads to exchange of adhesion proteins, which are replaced by adaptor proteins (e.g., clathrin and Dab2) for integrin endocytosis ( Yu et al. , 2015 ).
Figure Legend Snippet: Switchblade model for integrin activation (adapted from ) and schematic presenting the hypothesis that cis interaction of α IIb β 3 integrin and RGD P2Y 2 R mediates integrin activation initiated by binding of SNV to the PSI domain. (A) 1) Structure of an inactive integrin. 2–3) Intracellular signaling (inside-out) induces integrin activation mediated by binding of adaptor proteins (such as talin) to the extended conformation with open head-piece bound to soluble and immobilized ligands (see the text for details). 4) Development of mechanochemical force selectively transduced through the β subunit. Integrin binding to immobilized ligand resists lateral translation and causes an increase in force (indicated by arrows) and promotes separation of the α− and β-subunit transmembrane domains. (B) 5) P2Y 2 R interacts in cis with α IIb β 3 integrin. 6) SNV occupancy of the PSI domain induces an increase in integrin affinity for cis - RGD P2Y 2 R (based on AFM measurements). Integrin extension exerts a membrane normal/lateral pulling force, due to height differences between the membrane proximal RGD P2Y 2 R, and the unbending integrin ( Takagi et al. , 2002 ; Chigaev et al. , 2003, 2015); Outside-in signaling. 7) Recruitment of talin and other adhesion molecules increases force transduction, which is terminated by rupture of RGD interaction. Full extension of α IIb β 3 integrin causes rupture of cis interaction, indicated by PAC1 staining of cells. 8) Cessation of tensile force, and loss of intracellular link to actin, leads to exchange of adhesion proteins, which are replaced by adaptor proteins (e.g., clathrin and Dab2) for integrin endocytosis ( Yu et al. , 2015 ).

Techniques Used: Activation Assay, Binding Assay, Transduction, Staining

P2Y 2 R expression in various cell lines. (A) Plot of P2ry2 mRNA expression in cell lines used in this study, namely P2Y 2 R-null wild type astrocytoma cells (WT1321N1), 1321N1 cells stably expressing an Arg95-Gly96-Glu97 (RGE) mutation of the Arg95-Gly96-Asp97 (RGD) sequence in the P2Y 2 R ( RGE P2Y 2 R) and 1321N1 cells expressing wild-type P2Y 2 R ( RGD P2Y 2 R), CHO-K1 and telomerase-immortalized human microvascular endothelium cell line (TIME). RNA was extracted from 150,000 cells in duplicate wells with RNeasy Qiagen kit. Quantitative RT–PCR was performed in triplicate for each well by Taqman assay as described under Materials and Methods . (B) Plot of P2ry2 mRNA knockdown in CHO-K1 stably transfected with α IIb β 3 –integrin measured 24 h after siRNA transfection, ** p < 0.05.
Figure Legend Snippet: P2Y 2 R expression in various cell lines. (A) Plot of P2ry2 mRNA expression in cell lines used in this study, namely P2Y 2 R-null wild type astrocytoma cells (WT1321N1), 1321N1 cells stably expressing an Arg95-Gly96-Glu97 (RGE) mutation of the Arg95-Gly96-Asp97 (RGD) sequence in the P2Y 2 R ( RGE P2Y 2 R) and 1321N1 cells expressing wild-type P2Y 2 R ( RGD P2Y 2 R), CHO-K1 and telomerase-immortalized human microvascular endothelium cell line (TIME). RNA was extracted from 150,000 cells in duplicate wells with RNeasy Qiagen kit. Quantitative RT–PCR was performed in triplicate for each well by Taqman assay as described under Materials and Methods . (B) Plot of P2ry2 mRNA knockdown in CHO-K1 stably transfected with α IIb β 3 –integrin measured 24 h after siRNA transfection, ** p < 0.05.

Techniques Used: Expressing, Stable Transfection, Mutagenesis, Sequencing, Quantitative RT-PCR, TaqMan Assay, Transfection

Experimental setup for single-molecule force microscopy of α IIb β 3 –RGD interaction. (A) Schematic and bottom view microgragh of AFM microcantilever functionalized with α IIb β 3 integrin above RGD P2Y 2 R binding sites on cell membranes. For the 1321N1 astrocytes, experiments were performed in DMEM containing 5% FBS with addition of 10 mM HEPES (pH 7.4). For CHO-K1, we used Tyrode’s buffer (Sigma-Aldrich) containing 1 mM CaCl 2 , 1 mM MgCl 2 , 0.1% glucose, and 0.1% BSA. For Mn 2+ activation, 1 mM CaCl 2 and 1 mM MgCl 2 were replaced with 2 mM MnCl 2 . (B) A typical cantilever- RGD P2Y 2 R 1321N1 force scan. Gray and black traces are engagement and retract traces, receptively. F u is the unbinding force on the retract trace. The lower panel shows the four stages of stretching and rupturing a single ligand-receptor complex using the AFM: 1) AFM-cell surface engagement, 2) retraction, 3) extension, and 4) rupture.
Figure Legend Snippet: Experimental setup for single-molecule force microscopy of α IIb β 3 –RGD interaction. (A) Schematic and bottom view microgragh of AFM microcantilever functionalized with α IIb β 3 integrin above RGD P2Y 2 R binding sites on cell membranes. For the 1321N1 astrocytes, experiments were performed in DMEM containing 5% FBS with addition of 10 mM HEPES (pH 7.4). For CHO-K1, we used Tyrode’s buffer (Sigma-Aldrich) containing 1 mM CaCl 2 , 1 mM MgCl 2 , 0.1% glucose, and 0.1% BSA. For Mn 2+ activation, 1 mM CaCl 2 and 1 mM MgCl 2 were replaced with 2 mM MnCl 2 . (B) A typical cantilever- RGD P2Y 2 R 1321N1 force scan. Gray and black traces are engagement and retract traces, receptively. F u is the unbinding force on the retract trace. The lower panel shows the four stages of stretching and rupturing a single ligand-receptor complex using the AFM: 1) AFM-cell surface engagement, 2) retraction, 3) extension, and 4) rupture.

Techniques Used: Microscopy, Binding Assay, Activation Assay

The frequency of adhesive interactions between an α IIb β 3 -functionalized AFM tip and cell surfaces increase with exogenous RGD P2Y 2 R and not RGE P2Y 2 R. (A) Adhesion frequency of α IIb β 3 -functionalized AFM cell membranes of P2Y 2 R-null WT, RGE P2Y 2 R, and RGD P2Y 2 R 1321N1 cells under similar AFM scan conditions. Values are means ± SD of force scans of greater than or equal to five different cell measurements. Statistical significance was determined by Student’s t test **** p < 0.0001. (B–D) Representative single force distance (retraction) curves between α IIb β 3 -functionalized AFM tip and the surface of parent WT 1321N1 cells devoid of P2Y 2 R, and 1321N1 cells expressing exogenous RGE P2Y 2 R and RGD P2Y 2 R. The fractions shown represent the number of positive force scans in the numerator and the denominator, the total number of scans for each cell type.
Figure Legend Snippet: The frequency of adhesive interactions between an α IIb β 3 -functionalized AFM tip and cell surfaces increase with exogenous RGD P2Y 2 R and not RGE P2Y 2 R. (A) Adhesion frequency of α IIb β 3 -functionalized AFM cell membranes of P2Y 2 R-null WT, RGE P2Y 2 R, and RGD P2Y 2 R 1321N1 cells under similar AFM scan conditions. Values are means ± SD of force scans of greater than or equal to five different cell measurements. Statistical significance was determined by Student’s t test **** p < 0.0001. (B–D) Representative single force distance (retraction) curves between α IIb β 3 -functionalized AFM tip and the surface of parent WT 1321N1 cells devoid of P2Y 2 R, and 1321N1 cells expressing exogenous RGE P2Y 2 R and RGD P2Y 2 R. The fractions shown represent the number of positive force scans in the numerator and the denominator, the total number of scans for each cell type.

Techniques Used: Expressing

Distributions of single molecule unbinding force measured under the same conditions of contact force retraction time and pulling speed. Force histograms show nonspecific interactions at P2Y2R-null WT and RGE P2Y 2 R 1321N1 cell membranes and RGD specific interactions at RGD P2Y 2 R 1321N1 cell membranes. (A) Force histograms of unitary nonspecific interactions between α IIb β 3 and WT 1321N1 cell membranes (the loading rate, r f , was 880 pN/s). Nonspecific adhesions on five separate cells, and the composite histogram of all force scans are shown. The fractions shown in each panel represent adhesions (numerator) and total number of force distance measurements (denominator). (B) Force histograms of RGE P2Y 2 R 1321N1 cell membranes measured under similar conditions to WT ( r f was 897 pN/s). (C) Force histograms of RGD P2Y 2 R 1321N1 cell membranes ( r f was 930 pN/s). The composite histogram shows a distribution of nonspecific (33% of total events) and RGD specific (66% of total events). The mode of the nonspecific adhesions is similar to WT and RGE.
Figure Legend Snippet: Distributions of single molecule unbinding force measured under the same conditions of contact force retraction time and pulling speed. Force histograms show nonspecific interactions at P2Y2R-null WT and RGE P2Y 2 R 1321N1 cell membranes and RGD specific interactions at RGD P2Y 2 R 1321N1 cell membranes. (A) Force histograms of unitary nonspecific interactions between α IIb β 3 and WT 1321N1 cell membranes (the loading rate, r f , was 880 pN/s). Nonspecific adhesions on five separate cells, and the composite histogram of all force scans are shown. The fractions shown in each panel represent adhesions (numerator) and total number of force distance measurements (denominator). (B) Force histograms of RGE P2Y 2 R 1321N1 cell membranes measured under similar conditions to WT ( r f was 897 pN/s). (C) Force histograms of RGD P2Y 2 R 1321N1 cell membranes ( r f was 930 pN/s). The composite histogram shows a distribution of nonspecific (33% of total events) and RGD specific (66% of total events). The mode of the nonspecific adhesions is similar to WT and RGE.

Techniques Used:

AFM measurements of unitary α IIb β 3 - RGD P2Y 2 R interactions performed under various conditions in WT CHO-K1 cells that are devoid of β 3 integrins. Measurements of SNV-integrin (PSI) interaction measured in CHO-A24 cells stably expressing α IIb β 3 are included. (A) The measurements were performed with an adhesion frequency of ∼33%. Shown are 10 representative consecutive force-distance (retraction) traces between a cantilever tip functionalized with α IIb β 3 -integrin and a CHO-K1 cell. The 4th, 6th and 9th force curves reveal unitary RGD-specific adhesive interactions. The 7th force curve shows typical nonspecific interactions, which were present with the heterobifunctional acetal-PEG27-NHS linker (PEG) only. These weak adhesions were ∼6% of the adhesion frequency events as shown in B. (B) Adhesion frequency measurements under various conditions: PEG was used to measure nonspecific interactions. siRNA refers to cells treated with P2Y 2 R siRNA 24 h prior to the experiment. Experiments were performed in Tyrode’s buffer (Sigma-Aldrich) containing 1 mM CaCl 2 , 1 mM MgCl 2 , 0.1% glucose, and 0.1% BSA. For Mn 2+ activation, CaCl 2 and MgCl 2 were replaced with 2 mM MnCl 2 . For SNV assays, the cantilever was preincubated with fluorescently labeled neat SNV R18 or a mixture of SNV R18 and 25 µM PSI domain polypeptide to competitively block the interaction between the integrin functionalized cantilever and SNV R18 . The AFM cantilever was washed before immersion into the sample chamber. Association of SNV and the integrin-functionalized AFM tip was confirmed by imaging SNV fluorescence on the AFM tip. Error bars represent SEM for greater than or equal to five separate measures such as shown in . * p < 0.05.
Figure Legend Snippet: AFM measurements of unitary α IIb β 3 - RGD P2Y 2 R interactions performed under various conditions in WT CHO-K1 cells that are devoid of β 3 integrins. Measurements of SNV-integrin (PSI) interaction measured in CHO-A24 cells stably expressing α IIb β 3 are included. (A) The measurements were performed with an adhesion frequency of ∼33%. Shown are 10 representative consecutive force-distance (retraction) traces between a cantilever tip functionalized with α IIb β 3 -integrin and a CHO-K1 cell. The 4th, 6th and 9th force curves reveal unitary RGD-specific adhesive interactions. The 7th force curve shows typical nonspecific interactions, which were present with the heterobifunctional acetal-PEG27-NHS linker (PEG) only. These weak adhesions were ∼6% of the adhesion frequency events as shown in B. (B) Adhesion frequency measurements under various conditions: PEG was used to measure nonspecific interactions. siRNA refers to cells treated with P2Y 2 R siRNA 24 h prior to the experiment. Experiments were performed in Tyrode’s buffer (Sigma-Aldrich) containing 1 mM CaCl 2 , 1 mM MgCl 2 , 0.1% glucose, and 0.1% BSA. For Mn 2+ activation, CaCl 2 and MgCl 2 were replaced with 2 mM MnCl 2 . For SNV assays, the cantilever was preincubated with fluorescently labeled neat SNV R18 or a mixture of SNV R18 and 25 µM PSI domain polypeptide to competitively block the interaction between the integrin functionalized cantilever and SNV R18 . The AFM cantilever was washed before immersion into the sample chamber. Association of SNV and the integrin-functionalized AFM tip was confirmed by imaging SNV fluorescence on the AFM tip. Error bars represent SEM for greater than or equal to five separate measures such as shown in . * p < 0.05.

Techniques Used: Stable Transfection, Expressing, Activation Assay, Labeling, Blocking Assay, Imaging, Fluorescence

Binding of SNV to integrin PSI induces integrin activation. (A) Force histogram of low affinity α IIb β 3 - RGD P2Y 2 R interactions; (the loading rate, r f , was 720 pN/s; N refers to the total number of adhesion events for all histograms). (B) Force histogram of α IIb β 3 - RGD P2Y 2 R interactions in the presence of 2 mM Mn 2+ ( r f = 700 pN/s). (C) Fluorescence micrographs (bottom view) of an integrin-functionalized cantilever preincubated with fluorescently labeled SNV R18 . (D) Force histogram of α IIb β 3 - RGD P2Y 2 R interactions with SNV bound to the integrin PSI domain ( r f = 739 pN/s). (E) Fluorescence micrographs (bottom view) of an integrin-functionalized cantilever preincubated with fluorescently labeled SNV R18 and PSI domain polypeptide for blocking SNV binding. (F) Force histogram of integrin after probe was exposed to SNV R18 and PSI domain peptide. (G) SNV functionalized cantilever. (H) Force histogram of SNV–α IIb β 3 interactions in CHO-A24 cells.
Figure Legend Snippet: Binding of SNV to integrin PSI induces integrin activation. (A) Force histogram of low affinity α IIb β 3 - RGD P2Y 2 R interactions; (the loading rate, r f , was 720 pN/s; N refers to the total number of adhesion events for all histograms). (B) Force histogram of α IIb β 3 - RGD P2Y 2 R interactions in the presence of 2 mM Mn 2+ ( r f = 700 pN/s). (C) Fluorescence micrographs (bottom view) of an integrin-functionalized cantilever preincubated with fluorescently labeled SNV R18 . (D) Force histogram of α IIb β 3 - RGD P2Y 2 R interactions with SNV bound to the integrin PSI domain ( r f = 739 pN/s). (E) Fluorescence micrographs (bottom view) of an integrin-functionalized cantilever preincubated with fluorescently labeled SNV R18 and PSI domain polypeptide for blocking SNV binding. (F) Force histogram of integrin after probe was exposed to SNV R18 and PSI domain peptide. (G) SNV functionalized cantilever. (H) Force histogram of SNV–α IIb β 3 interactions in CHO-A24 cells.

Techniques Used: Binding Assay, Activation Assay, Fluorescence, Labeling, Blocking Assay

Analysis of potential barrier energy landscapes and bond lifetimes by dynamic force spectroscopy (A) Dynamic force spectrum: Plot of most probable unbinding force ( f * in Eq. 2) of α IIB β 3 –integrin/RGD interactions vs. logarithm of loading rate, experimental data are shown as follows: red square (SNV activated integrin), blue circle (Mn 2+ -activated integrin), and open circle (low affinity integrin). Lines represent fits to Eq. 2. γ, the distance of the potential barrier position from the bottom of the potential well, is derived from the slope. The force-free dissociation rate, k 0 , is derived from the intercept of the fit. (B) Potential energy landscape of the α IIB β 3 – RGD P2Y 2 R bond qualitatively showing the relative magnitudes of energy barrier widths of SNV (0.35 nm) and Mn 2+ - (0.40 nm) activated interactions derived from the DFS. k 0 values for SNV and Mn 2+ -associated interactions were 0.13 s –1 and 0.097 s –1 , respectively. (C) Effect of tensile force (f in Eq. 1) on the lifetime of the α IIB β 3 - RGD P2Y 2 R interactions, for control and SNV- and Mn 2+ -treated samples. (D) Effect of tensile force on the potential energy barrier landscape of SNV- and Mn 2+ -treated integrin– RGD P2Y 2 R interactions (see the text for details). The mechanical energy for each applied force lowers the transition state energy barrier by -f•γ as shown.
Figure Legend Snippet: Analysis of potential barrier energy landscapes and bond lifetimes by dynamic force spectroscopy (A) Dynamic force spectrum: Plot of most probable unbinding force ( f * in Eq. 2) of α IIB β 3 –integrin/RGD interactions vs. logarithm of loading rate, experimental data are shown as follows: red square (SNV activated integrin), blue circle (Mn 2+ -activated integrin), and open circle (low affinity integrin). Lines represent fits to Eq. 2. γ, the distance of the potential barrier position from the bottom of the potential well, is derived from the slope. The force-free dissociation rate, k 0 , is derived from the intercept of the fit. (B) Potential energy landscape of the α IIB β 3 – RGD P2Y 2 R bond qualitatively showing the relative magnitudes of energy barrier widths of SNV (0.35 nm) and Mn 2+ - (0.40 nm) activated interactions derived from the DFS. k 0 values for SNV and Mn 2+ -associated interactions were 0.13 s –1 and 0.097 s –1 , respectively. (C) Effect of tensile force (f in Eq. 1) on the lifetime of the α IIB β 3 - RGD P2Y 2 R interactions, for control and SNV- and Mn 2+ -treated samples. (D) Effect of tensile force on the potential energy barrier landscape of SNV- and Mn 2+ -treated integrin– RGD P2Y 2 R interactions (see the text for details). The mechanical energy for each applied force lowers the transition state energy barrier by -f•γ as shown.

Techniques Used: Spectroscopy, Derivative Assay

SNV binding to integrin PSI domain recapitulates physiological activation of α Ilb β 3 . (A, B) Model of SNV induced extended conformation associated with a high-affinity state with separated α and β transmembrane and cytoplasmic domains, which allows the ligand mimetic monoclonal anti-α Ilb β 3 antibody PAC1 to bind to CHO-A24 cells stably expressing wild-type α IIb β 3 . Flow cytometry histogram inserts show fluorescence associated with (a) PAC-1 staining of 50,000 cells activated with SNV particles, (b) staining of resting cells, and (c) autofluorescence of unstained cells. The graph is a plot of PAC-1 staining (1:20 dilution) of CHO-A24 corrected for autofluorescence. Cells were exposed to SNV for 5 min at 37°C and then quenched on ice, fixed with 3% paraformaldehyde for 10 min, and stained with PAC1 at 1:20 dil. for 30 min on shaker at 37°C. Poor PAC1 staining of CHO-C3 cells expressing integrin mutant with an inner membrane clasp at the α IIb W968C/β3I693, which prevents subunit separation. Experiments were performed in parallel with CHO-A24. Error bar is SE for duplicate measurements, * p < 0.05. (C) Plot of fold increase over resting cells in PAC-1 staining of CHO-A24 cells in suspension after 30 min exposure to killed SNV. 10,000 CHO-A24 cells suspended in 20 µl media containing 1:10 dilution PAC-1 antibody, coadministered with 1 µM GRGDSP. (D) Titration of soluble GRGDSP peptide, which abrogates cis -interaction of the integrin and RGD P2Y 2 R, inhibits infection of suspension CHO-24 cells. CHO-A24 cells were infected in suspension with 0.1 moi SNV for 30 min. Cells were then washed in low pH media three times and then incubated in normal media for 24 h and then assayed for viral N-protein.
Figure Legend Snippet: SNV binding to integrin PSI domain recapitulates physiological activation of α Ilb β 3 . (A, B) Model of SNV induced extended conformation associated with a high-affinity state with separated α and β transmembrane and cytoplasmic domains, which allows the ligand mimetic monoclonal anti-α Ilb β 3 antibody PAC1 to bind to CHO-A24 cells stably expressing wild-type α IIb β 3 . Flow cytometry histogram inserts show fluorescence associated with (a) PAC-1 staining of 50,000 cells activated with SNV particles, (b) staining of resting cells, and (c) autofluorescence of unstained cells. The graph is a plot of PAC-1 staining (1:20 dilution) of CHO-A24 corrected for autofluorescence. Cells were exposed to SNV for 5 min at 37°C and then quenched on ice, fixed with 3% paraformaldehyde for 10 min, and stained with PAC1 at 1:20 dil. for 30 min on shaker at 37°C. Poor PAC1 staining of CHO-C3 cells expressing integrin mutant with an inner membrane clasp at the α IIb W968C/β3I693, which prevents subunit separation. Experiments were performed in parallel with CHO-A24. Error bar is SE for duplicate measurements, * p < 0.05. (C) Plot of fold increase over resting cells in PAC-1 staining of CHO-A24 cells in suspension after 30 min exposure to killed SNV. 10,000 CHO-A24 cells suspended in 20 µl media containing 1:10 dilution PAC-1 antibody, coadministered with 1 µM GRGDSP. (D) Titration of soluble GRGDSP peptide, which abrogates cis -interaction of the integrin and RGD P2Y 2 R, inhibits infection of suspension CHO-24 cells. CHO-A24 cells were infected in suspension with 0.1 moi SNV for 30 min. Cells were then washed in low pH media three times and then incubated in normal media for 24 h and then assayed for viral N-protein.

Techniques Used: Binding Assay, Activation Assay, Stable Transfection, Expressing, Flow Cytometry, Fluorescence, Staining, Mutagenesis, Titration, Infection, Incubation

Cell polarity disrupts the cis interaction between P2Y 2 R and β 3 integrin and infectivity. (A) Confocal image of polarized Vero E6 cells expressing antibody-stained basolateral α v β 3 and apical P2Y 2 R. The top panel is a confocal image in which the focus plane was parallel to the monolayer ( XY scan), whereas the bottom panel shows the focus plane as a vertical cross section of the monolayer ( XZ scan). The white line indicated by the arrow in the XY scan indicates the path of the XZ scan. To enable visualization, Vero E6 cells were transiently transfected with P2Y 2 R and plated at confluence in eight-well Nunc Lab-Tek chambers and allowed to propagate for 1–2 d. At room temperature, cells were fixed with 3% paraformaldehyde for 20 min and subsequently permeabilized with 0.2% Triton X-100 (in PBS) for 15 min. Cells were blocked for nonspecific staining with 1% BSA in PBS for 30 min and then incubated with mouse monoclonal anti-integrin α v β 3 (1:40, Millipore MsxHu, MAB#1976) and rabbit polyclonal anti-P2Y 2 (1:100, Abcam, ab10270) were used with Alexa 488 and Alexa 580, respectively. (B) Plot of SNV FFU/ml vs. cells in suspension and on plates. CHO-A24, TIME, and Vero E6 cells were plated at confluence in vitronectin treated 48-well plates for 24 h. Confluent cells in triplicate wells were inoculated with 0.1 moi SNV for 1 h, washed in low pH media, and then incubated in normal media for 24 h. Media from infected cells was collected after 24 h diluted and subjected to a 7-day focus assay in Vero cells (see Supplemental Figure S1E for typical SNV FFU). ** p < 0.001.
Figure Legend Snippet: Cell polarity disrupts the cis interaction between P2Y 2 R and β 3 integrin and infectivity. (A) Confocal image of polarized Vero E6 cells expressing antibody-stained basolateral α v β 3 and apical P2Y 2 R. The top panel is a confocal image in which the focus plane was parallel to the monolayer ( XY scan), whereas the bottom panel shows the focus plane as a vertical cross section of the monolayer ( XZ scan). The white line indicated by the arrow in the XY scan indicates the path of the XZ scan. To enable visualization, Vero E6 cells were transiently transfected with P2Y 2 R and plated at confluence in eight-well Nunc Lab-Tek chambers and allowed to propagate for 1–2 d. At room temperature, cells were fixed with 3% paraformaldehyde for 20 min and subsequently permeabilized with 0.2% Triton X-100 (in PBS) for 15 min. Cells were blocked for nonspecific staining with 1% BSA in PBS for 30 min and then incubated with mouse monoclonal anti-integrin α v β 3 (1:40, Millipore MsxHu, MAB#1976) and rabbit polyclonal anti-P2Y 2 (1:100, Abcam, ab10270) were used with Alexa 488 and Alexa 580, respectively. (B) Plot of SNV FFU/ml vs. cells in suspension and on plates. CHO-A24, TIME, and Vero E6 cells were plated at confluence in vitronectin treated 48-well plates for 24 h. Confluent cells in triplicate wells were inoculated with 0.1 moi SNV for 1 h, washed in low pH media, and then incubated in normal media for 24 h. Media from infected cells was collected after 24 h diluted and subjected to a 7-day focus assay in Vero cells (see Supplemental Figure S1E for typical SNV FFU). ** p < 0.001.

Techniques Used: Infection, Expressing, Staining, Transfection, Incubation

Signaling modulation assays.
Figure Legend Snippet: Signaling modulation assays.

Techniques Used: Concentration Assay, Incubation


Structured Review

Santa Cruz Biotechnology p2y 2 r sirna
Switchblade model for integrin activation (adapted from ) and schematic presenting the hypothesis that cis interaction of α IIb β 3 integrin and RGD <t>P2Y</t> <t>2</t> <t>R</t> mediates integrin activation initiated by binding of SNV to the PSI domain. (A) 1) Structure of an inactive integrin. 2–3) Intracellular signaling (inside-out) induces integrin activation mediated by binding of adaptor proteins (such as talin) to the extended conformation with open head-piece bound to soluble and immobilized ligands (see the text for details). 4) Development of mechanochemical force selectively transduced through the β subunit. Integrin binding to immobilized ligand resists lateral translation and causes an increase in force (indicated by arrows) and promotes separation of the α− and β-subunit transmembrane domains. (B) 5) P2Y 2 R interacts in cis with α IIb β 3 integrin. 6) SNV occupancy of the PSI domain induces an increase in integrin affinity for cis - RGD P2Y 2 R (based on AFM measurements). Integrin extension exerts a membrane normal/lateral pulling force, due to height differences between the membrane proximal RGD P2Y 2 R, and the unbending integrin ( Takagi et al. , 2002 ; Chigaev et al. , 2003, 2015); Outside-in signaling. 7) Recruitment of talin and other adhesion molecules increases force transduction, which is terminated by rupture of RGD interaction. Full extension of α IIb β 3 integrin causes rupture of cis interaction, indicated by PAC1 staining of cells. 8) Cessation of tensile force, and loss of intracellular link to actin, leads to exchange of adhesion proteins, which are replaced by adaptor proteins (e.g., clathrin and Dab2) for integrin endocytosis ( Yu et al. , 2015 ).
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1) Product Images from "Low-affinity binding in cis to P2Y 2 R mediates force-dependent integrin activation during hantavirus infection"

Article Title: Low-affinity binding in cis to P2Y 2 R mediates force-dependent integrin activation during hantavirus infection

Journal: Molecular Biology of the Cell

doi: 10.1091/mbc.E17-01-0082

Switchblade model for integrin activation (adapted from ) and schematic presenting the hypothesis that cis interaction of α IIb β 3 integrin and RGD P2Y 2 R mediates integrin activation initiated by binding of SNV to the PSI domain. (A) 1) Structure of an inactive integrin. 2–3) Intracellular signaling (inside-out) induces integrin activation mediated by binding of adaptor proteins (such as talin) to the extended conformation with open head-piece bound to soluble and immobilized ligands (see the text for details). 4) Development of mechanochemical force selectively transduced through the β subunit. Integrin binding to immobilized ligand resists lateral translation and causes an increase in force (indicated by arrows) and promotes separation of the α− and β-subunit transmembrane domains. (B) 5) P2Y 2 R interacts in cis with α IIb β 3 integrin. 6) SNV occupancy of the PSI domain induces an increase in integrin affinity for cis - RGD P2Y 2 R (based on AFM measurements). Integrin extension exerts a membrane normal/lateral pulling force, due to height differences between the membrane proximal RGD P2Y 2 R, and the unbending integrin ( Takagi et al. , 2002 ; Chigaev et al. , 2003, 2015); Outside-in signaling. 7) Recruitment of talin and other adhesion molecules increases force transduction, which is terminated by rupture of RGD interaction. Full extension of α IIb β 3 integrin causes rupture of cis interaction, indicated by PAC1 staining of cells. 8) Cessation of tensile force, and loss of intracellular link to actin, leads to exchange of adhesion proteins, which are replaced by adaptor proteins (e.g., clathrin and Dab2) for integrin endocytosis ( Yu et al. , 2015 ).
Figure Legend Snippet: Switchblade model for integrin activation (adapted from ) and schematic presenting the hypothesis that cis interaction of α IIb β 3 integrin and RGD P2Y 2 R mediates integrin activation initiated by binding of SNV to the PSI domain. (A) 1) Structure of an inactive integrin. 2–3) Intracellular signaling (inside-out) induces integrin activation mediated by binding of adaptor proteins (such as talin) to the extended conformation with open head-piece bound to soluble and immobilized ligands (see the text for details). 4) Development of mechanochemical force selectively transduced through the β subunit. Integrin binding to immobilized ligand resists lateral translation and causes an increase in force (indicated by arrows) and promotes separation of the α− and β-subunit transmembrane domains. (B) 5) P2Y 2 R interacts in cis with α IIb β 3 integrin. 6) SNV occupancy of the PSI domain induces an increase in integrin affinity for cis - RGD P2Y 2 R (based on AFM measurements). Integrin extension exerts a membrane normal/lateral pulling force, due to height differences between the membrane proximal RGD P2Y 2 R, and the unbending integrin ( Takagi et al. , 2002 ; Chigaev et al. , 2003, 2015); Outside-in signaling. 7) Recruitment of talin and other adhesion molecules increases force transduction, which is terminated by rupture of RGD interaction. Full extension of α IIb β 3 integrin causes rupture of cis interaction, indicated by PAC1 staining of cells. 8) Cessation of tensile force, and loss of intracellular link to actin, leads to exchange of adhesion proteins, which are replaced by adaptor proteins (e.g., clathrin and Dab2) for integrin endocytosis ( Yu et al. , 2015 ).

Techniques Used: Activation Assay, Binding Assay, Transduction, Staining

P2Y 2 R expression in various cell lines. (A) Plot of P2ry2 mRNA expression in cell lines used in this study, namely P2Y 2 R-null wild type astrocytoma cells (WT1321N1), 1321N1 cells stably expressing an Arg95-Gly96-Glu97 (RGE) mutation of the Arg95-Gly96-Asp97 (RGD) sequence in the P2Y 2 R ( RGE P2Y 2 R) and 1321N1 cells expressing wild-type P2Y 2 R ( RGD P2Y 2 R), CHO-K1 and telomerase-immortalized human microvascular endothelium cell line (TIME). RNA was extracted from 150,000 cells in duplicate wells with RNeasy Qiagen kit. Quantitative RT–PCR was performed in triplicate for each well by Taqman assay as described under Materials and Methods . (B) Plot of P2ry2 mRNA knockdown in CHO-K1 stably transfected with α IIb β 3 –integrin measured 24 h after siRNA transfection, ** p < 0.05.
Figure Legend Snippet: P2Y 2 R expression in various cell lines. (A) Plot of P2ry2 mRNA expression in cell lines used in this study, namely P2Y 2 R-null wild type astrocytoma cells (WT1321N1), 1321N1 cells stably expressing an Arg95-Gly96-Glu97 (RGE) mutation of the Arg95-Gly96-Asp97 (RGD) sequence in the P2Y 2 R ( RGE P2Y 2 R) and 1321N1 cells expressing wild-type P2Y 2 R ( RGD P2Y 2 R), CHO-K1 and telomerase-immortalized human microvascular endothelium cell line (TIME). RNA was extracted from 150,000 cells in duplicate wells with RNeasy Qiagen kit. Quantitative RT–PCR was performed in triplicate for each well by Taqman assay as described under Materials and Methods . (B) Plot of P2ry2 mRNA knockdown in CHO-K1 stably transfected with α IIb β 3 –integrin measured 24 h after siRNA transfection, ** p < 0.05.

Techniques Used: Expressing, Stable Transfection, Mutagenesis, Sequencing, Quantitative RT-PCR, TaqMan Assay, Transfection

Experimental setup for single-molecule force microscopy of α IIb β 3 –RGD interaction. (A) Schematic and bottom view microgragh of AFM microcantilever functionalized with α IIb β 3 integrin above RGD P2Y 2 R binding sites on cell membranes. For the 1321N1 astrocytes, experiments were performed in DMEM containing 5% FBS with addition of 10 mM HEPES (pH 7.4). For CHO-K1, we used Tyrode’s buffer (Sigma-Aldrich) containing 1 mM CaCl 2 , 1 mM MgCl 2 , 0.1% glucose, and 0.1% BSA. For Mn 2+ activation, 1 mM CaCl 2 and 1 mM MgCl 2 were replaced with 2 mM MnCl 2 . (B) A typical cantilever- RGD P2Y 2 R 1321N1 force scan. Gray and black traces are engagement and retract traces, receptively. F u is the unbinding force on the retract trace. The lower panel shows the four stages of stretching and rupturing a single ligand-receptor complex using the AFM: 1) AFM-cell surface engagement, 2) retraction, 3) extension, and 4) rupture.
Figure Legend Snippet: Experimental setup for single-molecule force microscopy of α IIb β 3 –RGD interaction. (A) Schematic and bottom view microgragh of AFM microcantilever functionalized with α IIb β 3 integrin above RGD P2Y 2 R binding sites on cell membranes. For the 1321N1 astrocytes, experiments were performed in DMEM containing 5% FBS with addition of 10 mM HEPES (pH 7.4). For CHO-K1, we used Tyrode’s buffer (Sigma-Aldrich) containing 1 mM CaCl 2 , 1 mM MgCl 2 , 0.1% glucose, and 0.1% BSA. For Mn 2+ activation, 1 mM CaCl 2 and 1 mM MgCl 2 were replaced with 2 mM MnCl 2 . (B) A typical cantilever- RGD P2Y 2 R 1321N1 force scan. Gray and black traces are engagement and retract traces, receptively. F u is the unbinding force on the retract trace. The lower panel shows the four stages of stretching and rupturing a single ligand-receptor complex using the AFM: 1) AFM-cell surface engagement, 2) retraction, 3) extension, and 4) rupture.

Techniques Used: Microscopy, Binding Assay, Activation Assay

The frequency of adhesive interactions between an α IIb β 3 -functionalized AFM tip and cell surfaces increase with exogenous RGD P2Y 2 R and not RGE P2Y 2 R. (A) Adhesion frequency of α IIb β 3 -functionalized AFM cell membranes of P2Y 2 R-null WT, RGE P2Y 2 R, and RGD P2Y 2 R 1321N1 cells under similar AFM scan conditions. Values are means ± SD of force scans of greater than or equal to five different cell measurements. Statistical significance was determined by Student’s t test **** p < 0.0001. (B–D) Representative single force distance (retraction) curves between α IIb β 3 -functionalized AFM tip and the surface of parent WT 1321N1 cells devoid of P2Y 2 R, and 1321N1 cells expressing exogenous RGE P2Y 2 R and RGD P2Y 2 R. The fractions shown represent the number of positive force scans in the numerator and the denominator, the total number of scans for each cell type.
Figure Legend Snippet: The frequency of adhesive interactions between an α IIb β 3 -functionalized AFM tip and cell surfaces increase with exogenous RGD P2Y 2 R and not RGE P2Y 2 R. (A) Adhesion frequency of α IIb β 3 -functionalized AFM cell membranes of P2Y 2 R-null WT, RGE P2Y 2 R, and RGD P2Y 2 R 1321N1 cells under similar AFM scan conditions. Values are means ± SD of force scans of greater than or equal to five different cell measurements. Statistical significance was determined by Student’s t test **** p < 0.0001. (B–D) Representative single force distance (retraction) curves between α IIb β 3 -functionalized AFM tip and the surface of parent WT 1321N1 cells devoid of P2Y 2 R, and 1321N1 cells expressing exogenous RGE P2Y 2 R and RGD P2Y 2 R. The fractions shown represent the number of positive force scans in the numerator and the denominator, the total number of scans for each cell type.

Techniques Used: Expressing

Distributions of single molecule unbinding force measured under the same conditions of contact force retraction time and pulling speed. Force histograms show nonspecific interactions at P2Y2R-null WT and RGE P2Y 2 R 1321N1 cell membranes and RGD specific interactions at RGD P2Y 2 R 1321N1 cell membranes. (A) Force histograms of unitary nonspecific interactions between α IIb β 3 and WT 1321N1 cell membranes (the loading rate, r f , was 880 pN/s). Nonspecific adhesions on five separate cells, and the composite histogram of all force scans are shown. The fractions shown in each panel represent adhesions (numerator) and total number of force distance measurements (denominator). (B) Force histograms of RGE P2Y 2 R 1321N1 cell membranes measured under similar conditions to WT ( r f was 897 pN/s). (C) Force histograms of RGD P2Y 2 R 1321N1 cell membranes ( r f was 930 pN/s). The composite histogram shows a distribution of nonspecific (33% of total events) and RGD specific (66% of total events). The mode of the nonspecific adhesions is similar to WT and RGE.
Figure Legend Snippet: Distributions of single molecule unbinding force measured under the same conditions of contact force retraction time and pulling speed. Force histograms show nonspecific interactions at P2Y2R-null WT and RGE P2Y 2 R 1321N1 cell membranes and RGD specific interactions at RGD P2Y 2 R 1321N1 cell membranes. (A) Force histograms of unitary nonspecific interactions between α IIb β 3 and WT 1321N1 cell membranes (the loading rate, r f , was 880 pN/s). Nonspecific adhesions on five separate cells, and the composite histogram of all force scans are shown. The fractions shown in each panel represent adhesions (numerator) and total number of force distance measurements (denominator). (B) Force histograms of RGE P2Y 2 R 1321N1 cell membranes measured under similar conditions to WT ( r f was 897 pN/s). (C) Force histograms of RGD P2Y 2 R 1321N1 cell membranes ( r f was 930 pN/s). The composite histogram shows a distribution of nonspecific (33% of total events) and RGD specific (66% of total events). The mode of the nonspecific adhesions is similar to WT and RGE.

Techniques Used:

AFM measurements of unitary α IIb β 3 - RGD P2Y 2 R interactions performed under various conditions in WT CHO-K1 cells that are devoid of β 3 integrins. Measurements of SNV-integrin (PSI) interaction measured in CHO-A24 cells stably expressing α IIb β 3 are included. (A) The measurements were performed with an adhesion frequency of ∼33%. Shown are 10 representative consecutive force-distance (retraction) traces between a cantilever tip functionalized with α IIb β 3 -integrin and a CHO-K1 cell. The 4th, 6th and 9th force curves reveal unitary RGD-specific adhesive interactions. The 7th force curve shows typical nonspecific interactions, which were present with the heterobifunctional acetal-PEG27-NHS linker (PEG) only. These weak adhesions were ∼6% of the adhesion frequency events as shown in B. (B) Adhesion frequency measurements under various conditions: PEG was used to measure nonspecific interactions. siRNA refers to cells treated with P2Y 2 R siRNA 24 h prior to the experiment. Experiments were performed in Tyrode’s buffer (Sigma-Aldrich) containing 1 mM CaCl 2 , 1 mM MgCl 2 , 0.1% glucose, and 0.1% BSA. For Mn 2+ activation, CaCl 2 and MgCl 2 were replaced with 2 mM MnCl 2 . For SNV assays, the cantilever was preincubated with fluorescently labeled neat SNV R18 or a mixture of SNV R18 and 25 µM PSI domain polypeptide to competitively block the interaction between the integrin functionalized cantilever and SNV R18 . The AFM cantilever was washed before immersion into the sample chamber. Association of SNV and the integrin-functionalized AFM tip was confirmed by imaging SNV fluorescence on the AFM tip. Error bars represent SEM for greater than or equal to five separate measures such as shown in . * p < 0.05.
Figure Legend Snippet: AFM measurements of unitary α IIb β 3 - RGD P2Y 2 R interactions performed under various conditions in WT CHO-K1 cells that are devoid of β 3 integrins. Measurements of SNV-integrin (PSI) interaction measured in CHO-A24 cells stably expressing α IIb β 3 are included. (A) The measurements were performed with an adhesion frequency of ∼33%. Shown are 10 representative consecutive force-distance (retraction) traces between a cantilever tip functionalized with α IIb β 3 -integrin and a CHO-K1 cell. The 4th, 6th and 9th force curves reveal unitary RGD-specific adhesive interactions. The 7th force curve shows typical nonspecific interactions, which were present with the heterobifunctional acetal-PEG27-NHS linker (PEG) only. These weak adhesions were ∼6% of the adhesion frequency events as shown in B. (B) Adhesion frequency measurements under various conditions: PEG was used to measure nonspecific interactions. siRNA refers to cells treated with P2Y 2 R siRNA 24 h prior to the experiment. Experiments were performed in Tyrode’s buffer (Sigma-Aldrich) containing 1 mM CaCl 2 , 1 mM MgCl 2 , 0.1% glucose, and 0.1% BSA. For Mn 2+ activation, CaCl 2 and MgCl 2 were replaced with 2 mM MnCl 2 . For SNV assays, the cantilever was preincubated with fluorescently labeled neat SNV R18 or a mixture of SNV R18 and 25 µM PSI domain polypeptide to competitively block the interaction between the integrin functionalized cantilever and SNV R18 . The AFM cantilever was washed before immersion into the sample chamber. Association of SNV and the integrin-functionalized AFM tip was confirmed by imaging SNV fluorescence on the AFM tip. Error bars represent SEM for greater than or equal to five separate measures such as shown in . * p < 0.05.

Techniques Used: Stable Transfection, Expressing, Activation Assay, Labeling, Blocking Assay, Imaging, Fluorescence

Binding of SNV to integrin PSI induces integrin activation. (A) Force histogram of low affinity α IIb β 3 - RGD P2Y 2 R interactions; (the loading rate, r f , was 720 pN/s; N refers to the total number of adhesion events for all histograms). (B) Force histogram of α IIb β 3 - RGD P2Y 2 R interactions in the presence of 2 mM Mn 2+ ( r f = 700 pN/s). (C) Fluorescence micrographs (bottom view) of an integrin-functionalized cantilever preincubated with fluorescently labeled SNV R18 . (D) Force histogram of α IIb β 3 - RGD P2Y 2 R interactions with SNV bound to the integrin PSI domain ( r f = 739 pN/s). (E) Fluorescence micrographs (bottom view) of an integrin-functionalized cantilever preincubated with fluorescently labeled SNV R18 and PSI domain polypeptide for blocking SNV binding. (F) Force histogram of integrin after probe was exposed to SNV R18 and PSI domain peptide. (G) SNV functionalized cantilever. (H) Force histogram of SNV–α IIb β 3 interactions in CHO-A24 cells.
Figure Legend Snippet: Binding of SNV to integrin PSI induces integrin activation. (A) Force histogram of low affinity α IIb β 3 - RGD P2Y 2 R interactions; (the loading rate, r f , was 720 pN/s; N refers to the total number of adhesion events for all histograms). (B) Force histogram of α IIb β 3 - RGD P2Y 2 R interactions in the presence of 2 mM Mn 2+ ( r f = 700 pN/s). (C) Fluorescence micrographs (bottom view) of an integrin-functionalized cantilever preincubated with fluorescently labeled SNV R18 . (D) Force histogram of α IIb β 3 - RGD P2Y 2 R interactions with SNV bound to the integrin PSI domain ( r f = 739 pN/s). (E) Fluorescence micrographs (bottom view) of an integrin-functionalized cantilever preincubated with fluorescently labeled SNV R18 and PSI domain polypeptide for blocking SNV binding. (F) Force histogram of integrin after probe was exposed to SNV R18 and PSI domain peptide. (G) SNV functionalized cantilever. (H) Force histogram of SNV–α IIb β 3 interactions in CHO-A24 cells.

Techniques Used: Binding Assay, Activation Assay, Fluorescence, Labeling, Blocking Assay

Analysis of potential barrier energy landscapes and bond lifetimes by dynamic force spectroscopy (A) Dynamic force spectrum: Plot of most probable unbinding force ( f * in Eq. 2) of α IIB β 3 –integrin/RGD interactions vs. logarithm of loading rate, experimental data are shown as follows: red square (SNV activated integrin), blue circle (Mn 2+ -activated integrin), and open circle (low affinity integrin). Lines represent fits to Eq. 2. γ, the distance of the potential barrier position from the bottom of the potential well, is derived from the slope. The force-free dissociation rate, k 0 , is derived from the intercept of the fit. (B) Potential energy landscape of the α IIB β 3 – RGD P2Y 2 R bond qualitatively showing the relative magnitudes of energy barrier widths of SNV (0.35 nm) and Mn 2+ - (0.40 nm) activated interactions derived from the DFS. k 0 values for SNV and Mn 2+ -associated interactions were 0.13 s –1 and 0.097 s –1 , respectively. (C) Effect of tensile force (f in Eq. 1) on the lifetime of the α IIB β 3 - RGD P2Y 2 R interactions, for control and SNV- and Mn 2+ -treated samples. (D) Effect of tensile force on the potential energy barrier landscape of SNV- and Mn 2+ -treated integrin– RGD P2Y 2 R interactions (see the text for details). The mechanical energy for each applied force lowers the transition state energy barrier by -f•γ as shown.
Figure Legend Snippet: Analysis of potential barrier energy landscapes and bond lifetimes by dynamic force spectroscopy (A) Dynamic force spectrum: Plot of most probable unbinding force ( f * in Eq. 2) of α IIB β 3 –integrin/RGD interactions vs. logarithm of loading rate, experimental data are shown as follows: red square (SNV activated integrin), blue circle (Mn 2+ -activated integrin), and open circle (low affinity integrin). Lines represent fits to Eq. 2. γ, the distance of the potential barrier position from the bottom of the potential well, is derived from the slope. The force-free dissociation rate, k 0 , is derived from the intercept of the fit. (B) Potential energy landscape of the α IIB β 3 – RGD P2Y 2 R bond qualitatively showing the relative magnitudes of energy barrier widths of SNV (0.35 nm) and Mn 2+ - (0.40 nm) activated interactions derived from the DFS. k 0 values for SNV and Mn 2+ -associated interactions were 0.13 s –1 and 0.097 s –1 , respectively. (C) Effect of tensile force (f in Eq. 1) on the lifetime of the α IIB β 3 - RGD P2Y 2 R interactions, for control and SNV- and Mn 2+ -treated samples. (D) Effect of tensile force on the potential energy barrier landscape of SNV- and Mn 2+ -treated integrin– RGD P2Y 2 R interactions (see the text for details). The mechanical energy for each applied force lowers the transition state energy barrier by -f•γ as shown.

Techniques Used: Spectroscopy, Derivative Assay

SNV binding to integrin PSI domain recapitulates physiological activation of α Ilb β 3 . (A, B) Model of SNV induced extended conformation associated with a high-affinity state with separated α and β transmembrane and cytoplasmic domains, which allows the ligand mimetic monoclonal anti-α Ilb β 3 antibody PAC1 to bind to CHO-A24 cells stably expressing wild-type α IIb β 3 . Flow cytometry histogram inserts show fluorescence associated with (a) PAC-1 staining of 50,000 cells activated with SNV particles, (b) staining of resting cells, and (c) autofluorescence of unstained cells. The graph is a plot of PAC-1 staining (1:20 dilution) of CHO-A24 corrected for autofluorescence. Cells were exposed to SNV for 5 min at 37°C and then quenched on ice, fixed with 3% paraformaldehyde for 10 min, and stained with PAC1 at 1:20 dil. for 30 min on shaker at 37°C. Poor PAC1 staining of CHO-C3 cells expressing integrin mutant with an inner membrane clasp at the α IIb W968C/β3I693, which prevents subunit separation. Experiments were performed in parallel with CHO-A24. Error bar is SE for duplicate measurements, * p < 0.05. (C) Plot of fold increase over resting cells in PAC-1 staining of CHO-A24 cells in suspension after 30 min exposure to killed SNV. 10,000 CHO-A24 cells suspended in 20 µl media containing 1:10 dilution PAC-1 antibody, coadministered with 1 µM GRGDSP. (D) Titration of soluble GRGDSP peptide, which abrogates cis -interaction of the integrin and RGD P2Y 2 R, inhibits infection of suspension CHO-24 cells. CHO-A24 cells were infected in suspension with 0.1 moi SNV for 30 min. Cells were then washed in low pH media three times and then incubated in normal media for 24 h and then assayed for viral N-protein.
Figure Legend Snippet: SNV binding to integrin PSI domain recapitulates physiological activation of α Ilb β 3 . (A, B) Model of SNV induced extended conformation associated with a high-affinity state with separated α and β transmembrane and cytoplasmic domains, which allows the ligand mimetic monoclonal anti-α Ilb β 3 antibody PAC1 to bind to CHO-A24 cells stably expressing wild-type α IIb β 3 . Flow cytometry histogram inserts show fluorescence associated with (a) PAC-1 staining of 50,000 cells activated with SNV particles, (b) staining of resting cells, and (c) autofluorescence of unstained cells. The graph is a plot of PAC-1 staining (1:20 dilution) of CHO-A24 corrected for autofluorescence. Cells were exposed to SNV for 5 min at 37°C and then quenched on ice, fixed with 3% paraformaldehyde for 10 min, and stained with PAC1 at 1:20 dil. for 30 min on shaker at 37°C. Poor PAC1 staining of CHO-C3 cells expressing integrin mutant with an inner membrane clasp at the α IIb W968C/β3I693, which prevents subunit separation. Experiments were performed in parallel with CHO-A24. Error bar is SE for duplicate measurements, * p < 0.05. (C) Plot of fold increase over resting cells in PAC-1 staining of CHO-A24 cells in suspension after 30 min exposure to killed SNV. 10,000 CHO-A24 cells suspended in 20 µl media containing 1:10 dilution PAC-1 antibody, coadministered with 1 µM GRGDSP. (D) Titration of soluble GRGDSP peptide, which abrogates cis -interaction of the integrin and RGD P2Y 2 R, inhibits infection of suspension CHO-24 cells. CHO-A24 cells were infected in suspension with 0.1 moi SNV for 30 min. Cells were then washed in low pH media three times and then incubated in normal media for 24 h and then assayed for viral N-protein.

Techniques Used: Binding Assay, Activation Assay, Stable Transfection, Expressing, Flow Cytometry, Fluorescence, Staining, Mutagenesis, Titration, Infection, Incubation

Cell polarity disrupts the cis interaction between P2Y 2 R and β 3 integrin and infectivity. (A) Confocal image of polarized Vero E6 cells expressing antibody-stained basolateral α v β 3 and apical P2Y 2 R. The top panel is a confocal image in which the focus plane was parallel to the monolayer ( XY scan), whereas the bottom panel shows the focus plane as a vertical cross section of the monolayer ( XZ scan). The white line indicated by the arrow in the XY scan indicates the path of the XZ scan. To enable visualization, Vero E6 cells were transiently transfected with P2Y 2 R and plated at confluence in eight-well Nunc Lab-Tek chambers and allowed to propagate for 1–2 d. At room temperature, cells were fixed with 3% paraformaldehyde for 20 min and subsequently permeabilized with 0.2% Triton X-100 (in PBS) for 15 min. Cells were blocked for nonspecific staining with 1% BSA in PBS for 30 min and then incubated with mouse monoclonal anti-integrin α v β 3 (1:40, Millipore MsxHu, MAB#1976) and rabbit polyclonal anti-P2Y 2 (1:100, Abcam, ab10270) were used with Alexa 488 and Alexa 580, respectively. (B) Plot of SNV FFU/ml vs. cells in suspension and on plates. CHO-A24, TIME, and Vero E6 cells were plated at confluence in vitronectin treated 48-well plates for 24 h. Confluent cells in triplicate wells were inoculated with 0.1 moi SNV for 1 h, washed in low pH media, and then incubated in normal media for 24 h. Media from infected cells was collected after 24 h diluted and subjected to a 7-day focus assay in Vero cells (see Supplemental Figure S1E for typical SNV FFU). ** p < 0.001.
Figure Legend Snippet: Cell polarity disrupts the cis interaction between P2Y 2 R and β 3 integrin and infectivity. (A) Confocal image of polarized Vero E6 cells expressing antibody-stained basolateral α v β 3 and apical P2Y 2 R. The top panel is a confocal image in which the focus plane was parallel to the monolayer ( XY scan), whereas the bottom panel shows the focus plane as a vertical cross section of the monolayer ( XZ scan). The white line indicated by the arrow in the XY scan indicates the path of the XZ scan. To enable visualization, Vero E6 cells were transiently transfected with P2Y 2 R and plated at confluence in eight-well Nunc Lab-Tek chambers and allowed to propagate for 1–2 d. At room temperature, cells were fixed with 3% paraformaldehyde for 20 min and subsequently permeabilized with 0.2% Triton X-100 (in PBS) for 15 min. Cells were blocked for nonspecific staining with 1% BSA in PBS for 30 min and then incubated with mouse monoclonal anti-integrin α v β 3 (1:40, Millipore MsxHu, MAB#1976) and rabbit polyclonal anti-P2Y 2 (1:100, Abcam, ab10270) were used with Alexa 488 and Alexa 580, respectively. (B) Plot of SNV FFU/ml vs. cells in suspension and on plates. CHO-A24, TIME, and Vero E6 cells were plated at confluence in vitronectin treated 48-well plates for 24 h. Confluent cells in triplicate wells were inoculated with 0.1 moi SNV for 1 h, washed in low pH media, and then incubated in normal media for 24 h. Media from infected cells was collected after 24 h diluted and subjected to a 7-day focus assay in Vero cells (see Supplemental Figure S1E for typical SNV FFU). ** p < 0.001.

Techniques Used: Infection, Expressing, Staining, Transfection, Incubation

Signaling modulation assays.
Figure Legend Snippet: Signaling modulation assays.

Techniques Used: Concentration Assay, Incubation


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Sirna Duplex Targeting P2y 2 R, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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