Structured Review

Proteintech antibodies against pdcd10
miR-222-3p directly suppresses <t>PDCD10</t> expression by binding to its 3' -UTR and inhibits EOC cell migration in vitro . ( A ) A Venn diagram was used to look for candidate genes targeted by miR-222-3p. ( B ) Levels of 15 candidate genes in HO 8910 PM cells transfected with miR-222-3p mimic. ( C ) miR-222-3p inhibitor assay in SKOV3 cells showing four candidate genes with upregulation of two genes, the most significant being PDCD10. ( D ) Western blot analysis of PDCD10 levels in two different EOC cell lines after transfection with miR-222-3p mimic or inhibitor. ( E ) Schematic depiction of the double-strand formation by miR-222-3p with the 3' -UTR of PDCD10. ( F ) Relative luciferase activities in HEK-293T cells co-transfected with a miR-222-3p mimic/inhibitor and PDCD10 WT/MUT. “++” stands for the double concentration. ( G and H ) Transwell and wound healing assays revealed inhibition of migration when HO 8910 PM cells were transfected with miR-222-3p mimic. Recovery assays showed that miR-222-3p suppressed migration of EOC cell lines due to its inhibitory effect on PDCD10 (Left). Cell counting and wound healing were quantified (Right). ( I ) qPCR and ( J ) Western blot analyses of PDCD10 expression levels in HO 8910 PM cells, and Image J calculated the relative expression rate. Data are means ± SEM. Data are from six (B) experiments and representative of three (C and F-J) independent experiments. *, P< 0.05; **, P< 0.01; ***, P< 0.001; ****, P< 0.0001, determined by unpaired two-tailed t-test.
Antibodies Against Pdcd10, supplied by Proteintech, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 93 stars, based on 1 article reviews
Price from $9.99 to $1999.99
antibodies against pdcd10 - by Bioz Stars, 2024-07
93/100 stars

Images

1) Product Images from "Non-canonical signaling pathway of SNAI2 induces EMT in ovarian cancer cells by suppressing miR-222-3p transcription and upregulating PDCD10"

Article Title: Non-canonical signaling pathway of SNAI2 induces EMT in ovarian cancer cells by suppressing miR-222-3p transcription and upregulating PDCD10

Journal: Theranostics

doi: 10.7150/thno.43198

miR-222-3p directly suppresses PDCD10 expression by binding to its 3' -UTR and inhibits EOC cell migration in vitro . ( A ) A Venn diagram was used to look for candidate genes targeted by miR-222-3p. ( B ) Levels of 15 candidate genes in HO 8910 PM cells transfected with miR-222-3p mimic. ( C ) miR-222-3p inhibitor assay in SKOV3 cells showing four candidate genes with upregulation of two genes, the most significant being PDCD10. ( D ) Western blot analysis of PDCD10 levels in two different EOC cell lines after transfection with miR-222-3p mimic or inhibitor. ( E ) Schematic depiction of the double-strand formation by miR-222-3p with the 3' -UTR of PDCD10. ( F ) Relative luciferase activities in HEK-293T cells co-transfected with a miR-222-3p mimic/inhibitor and PDCD10 WT/MUT. “++” stands for the double concentration. ( G and H ) Transwell and wound healing assays revealed inhibition of migration when HO 8910 PM cells were transfected with miR-222-3p mimic. Recovery assays showed that miR-222-3p suppressed migration of EOC cell lines due to its inhibitory effect on PDCD10 (Left). Cell counting and wound healing were quantified (Right). ( I ) qPCR and ( J ) Western blot analyses of PDCD10 expression levels in HO 8910 PM cells, and Image J calculated the relative expression rate. Data are means ± SEM. Data are from six (B) experiments and representative of three (C and F-J) independent experiments. *, P< 0.05; **, P< 0.01; ***, P< 0.001; ****, P< 0.0001, determined by unpaired two-tailed t-test.
Figure Legend Snippet: miR-222-3p directly suppresses PDCD10 expression by binding to its 3' -UTR and inhibits EOC cell migration in vitro . ( A ) A Venn diagram was used to look for candidate genes targeted by miR-222-3p. ( B ) Levels of 15 candidate genes in HO 8910 PM cells transfected with miR-222-3p mimic. ( C ) miR-222-3p inhibitor assay in SKOV3 cells showing four candidate genes with upregulation of two genes, the most significant being PDCD10. ( D ) Western blot analysis of PDCD10 levels in two different EOC cell lines after transfection with miR-222-3p mimic or inhibitor. ( E ) Schematic depiction of the double-strand formation by miR-222-3p with the 3' -UTR of PDCD10. ( F ) Relative luciferase activities in HEK-293T cells co-transfected with a miR-222-3p mimic/inhibitor and PDCD10 WT/MUT. “++” stands for the double concentration. ( G and H ) Transwell and wound healing assays revealed inhibition of migration when HO 8910 PM cells were transfected with miR-222-3p mimic. Recovery assays showed that miR-222-3p suppressed migration of EOC cell lines due to its inhibitory effect on PDCD10 (Left). Cell counting and wound healing were quantified (Right). ( I ) qPCR and ( J ) Western blot analyses of PDCD10 expression levels in HO 8910 PM cells, and Image J calculated the relative expression rate. Data are means ± SEM. Data are from six (B) experiments and representative of three (C and F-J) independent experiments. *, P< 0.05; **, P< 0.01; ***, P< 0.001; ****, P< 0.0001, determined by unpaired two-tailed t-test.

Techniques Used: Expressing, Binding Assay, Migration, In Vitro, Transfection, Western Blot, Luciferase, Concentration Assay, Inhibition, Cell Counting, Two Tailed Test

miR-222-3p suppresses EOC tumor metastasis in vivo by targeting PDCD10. ( A ) Schematic presentation of in vivo adhesion for equivalent numbers of GFP-labeled LV-miR-ctrl/LV-miR-222-3p and GFP-labeled ctrl vector/PDCD10 transfected stably in HO 8910 PM cells. Bar, 100 µm. ( B and C ) Representative images and quantification of intraperitoneal metastases in mice implanted intraperitoneally with the same number of HO 8910 PM cells (n= 4 mice per group). Bar, 1 cm. ( D ) Western blot analysis of PDCD10 levels in representative EOC metastatic nodules. ( E ) Representative images and bioluminescence mice images of lung tissue metastatic nodules 5 weeks (wk) after implantation. Bar, 0.5 cm. ( F ) Quantification of metastatic nodules in lung tissues of mice. ( G ) Representative images (Down) and metastatic nodule plots (Up) of mice stomach tissues formed in the restoration group. Bar, 0.5 cm. ( H ) IHC staining for PDCD10 in the stomach tissues of mice 5 weeks after implantation. Bar, 100 µm. ( I ) Representative images (Down) and metastatic nodule plots (Up) of mice liver tissues formed in the restoration group. Bar, 1 cm. ( J ) Representative HE staining of the mice lung tissues obtained from 5 weeks after implantation. Bar, 50 µm (Left) and 100 µm (Right). Data are means ± SEM and are representative of three (C, F, G, I) independent experiments. *, P< 0.05. **; P< 0.01; ***, P< 0.001, determined by unpaired two-tailed t-test.
Figure Legend Snippet: miR-222-3p suppresses EOC tumor metastasis in vivo by targeting PDCD10. ( A ) Schematic presentation of in vivo adhesion for equivalent numbers of GFP-labeled LV-miR-ctrl/LV-miR-222-3p and GFP-labeled ctrl vector/PDCD10 transfected stably in HO 8910 PM cells. Bar, 100 µm. ( B and C ) Representative images and quantification of intraperitoneal metastases in mice implanted intraperitoneally with the same number of HO 8910 PM cells (n= 4 mice per group). Bar, 1 cm. ( D ) Western blot analysis of PDCD10 levels in representative EOC metastatic nodules. ( E ) Representative images and bioluminescence mice images of lung tissue metastatic nodules 5 weeks (wk) after implantation. Bar, 0.5 cm. ( F ) Quantification of metastatic nodules in lung tissues of mice. ( G ) Representative images (Down) and metastatic nodule plots (Up) of mice stomach tissues formed in the restoration group. Bar, 0.5 cm. ( H ) IHC staining for PDCD10 in the stomach tissues of mice 5 weeks after implantation. Bar, 100 µm. ( I ) Representative images (Down) and metastatic nodule plots (Up) of mice liver tissues formed in the restoration group. Bar, 1 cm. ( J ) Representative HE staining of the mice lung tissues obtained from 5 weeks after implantation. Bar, 50 µm (Left) and 100 µm (Right). Data are means ± SEM and are representative of three (C, F, G, I) independent experiments. *, P< 0.05. **; P< 0.01; ***, P< 0.001, determined by unpaired two-tailed t-test.

Techniques Used: In Vivo, Labeling, Plasmid Preparation, Transfection, Stable Transfection, Western Blot, Immunohistochemistry, Staining, Two Tailed Test

miR-222-3p targets PDCD10 to inhibit cell migration via EMT pathway. ( A ) Meta-analysis describing forest plots of PDCD10 expression as a univariate predictor of overall survival. ( B ) Kaplan-Meier curves for overall survival probability in 1656 OC patients with low (n=640) and high (n=1016) PDCD10 expression (analyzed with log-rank test) from KMplot ( http://kmplot.com/analysis/ ). ( C ) qPCR and Western blot analyses of PDCD10 levels in FE25 and 4 different EOC cell lines. ( D ) Representative images of PDCD10 expression in normal and tumor ovary tissues. Bar, 100 µm (Left) and 30 µm (Right). ( E ) Enrichment evaluation within the PDCD10-expressing levels for predicted GSEA results of TCGA reference gene sets for high and low PDCD10 expression groups. Genes expressed in the profile datasets were ranked by fold changes (high-expression/low-expression). GSEA correlation pathways were determined by the given algorithm. Vertical bars along the x-axis of the GSEA diagram represent the positions within the ranked list of genes set in the given sets. Positive and negative GSEA curves mean positive and negative enrichments, respectively. ( F and G ) qPCR and Western blot analyses of PDCD10, E-cad (CDH1) and VIM expression in HO 8910 PM cells (PDCD10-overexpressing groups) and SKOV3 cells (PDCD10-silenced groups). ( H ) HO 8910 PM cells transduced with control vector or PDCD10 (GFP-labeled ctrl vector and PDCD10 all in green) were subjected to immunofluorescence with human-specific E-cad and VIM antibodies (in red). Bar, 10 µm. ( I ) Western blot analysis of PDCD10, E-cad, Vim translation levels in HO 8910 PM cells after treatment with miR-ctrl mimic or miR-222-3p mimic, in the presence or absence of PDCD10. Data are means ± SEM and are representative of three (C and F) independent experiments. *, P< 0.05; **, P< 0.01; ***, P< 0.001; ****, P< 0.0001, determined by unpaired two-tailed t-test.
Figure Legend Snippet: miR-222-3p targets PDCD10 to inhibit cell migration via EMT pathway. ( A ) Meta-analysis describing forest plots of PDCD10 expression as a univariate predictor of overall survival. ( B ) Kaplan-Meier curves for overall survival probability in 1656 OC patients with low (n=640) and high (n=1016) PDCD10 expression (analyzed with log-rank test) from KMplot ( http://kmplot.com/analysis/ ). ( C ) qPCR and Western blot analyses of PDCD10 levels in FE25 and 4 different EOC cell lines. ( D ) Representative images of PDCD10 expression in normal and tumor ovary tissues. Bar, 100 µm (Left) and 30 µm (Right). ( E ) Enrichment evaluation within the PDCD10-expressing levels for predicted GSEA results of TCGA reference gene sets for high and low PDCD10 expression groups. Genes expressed in the profile datasets were ranked by fold changes (high-expression/low-expression). GSEA correlation pathways were determined by the given algorithm. Vertical bars along the x-axis of the GSEA diagram represent the positions within the ranked list of genes set in the given sets. Positive and negative GSEA curves mean positive and negative enrichments, respectively. ( F and G ) qPCR and Western blot analyses of PDCD10, E-cad (CDH1) and VIM expression in HO 8910 PM cells (PDCD10-overexpressing groups) and SKOV3 cells (PDCD10-silenced groups). ( H ) HO 8910 PM cells transduced with control vector or PDCD10 (GFP-labeled ctrl vector and PDCD10 all in green) were subjected to immunofluorescence with human-specific E-cad and VIM antibodies (in red). Bar, 10 µm. ( I ) Western blot analysis of PDCD10, E-cad, Vim translation levels in HO 8910 PM cells after treatment with miR-ctrl mimic or miR-222-3p mimic, in the presence or absence of PDCD10. Data are means ± SEM and are representative of three (C and F) independent experiments. *, P< 0.05; **, P< 0.01; ***, P< 0.001; ****, P< 0.0001, determined by unpaired two-tailed t-test.

Techniques Used: Migration, Expressing, Western Blot, Transduction, Plasmid Preparation, Labeling, Immunofluorescence, Two Tailed Test

miR-222-3p targets PDCD10 to repress cell migration by downregulating the Wnt/ β -catenin signaling pathway. (A) qPCR analysis of β -catenin and TCF4 expression in HO 8910 PM cells (PDCD10-overexpressing groups) and SKOV3 cells (PDCD10-silenced groups). ( B ) Activity of the Wnt/ β -catenin signaling pathway in HEK-293T cells detected by the TOP flash/FOP flash dual-luciferase reporting system. HEK-293T cells were transfected with TOP flash/FOP flash plasmid and pRL-SV40 vector followed by transfection with or without PDCD10, and siRNA-NC/PDCD10, siRNA-03/PDCD10, or siRNA-04. Firefly luciferase activities were tested by a luminometer, and the activity of the Wnt/ β -catenin signaling pathway was recorded as TOP/FOP. ( C ) Western blot analysis of PDCD10 and β -catenin protein expression following overexpression of PDCD10 in HO 8910 PM cell fractions. β -actin was utilized as a marker for the cytosolic fractions. ( D ) HO 8910 PM cells transduced with or without PDCD10 were subjected to immunofluorescence with human-specific β -catenin antibodies. ( β -catenin in red). Bar, 10 µm. ( E ) β -catenin nuclei-intensity (detected by Operetta High-Content Imaging System (Perkin Elmer) in HO 8910 PM cell). ( F ) Western blot analysis of PDCD10, β -catenin translation levels in HO 8910 PM cells, or after coculturing with miR-ctrl mimic or miR-222-3p mimic in the presence or absence of PDCD10 (Left). The relative expression rate was calculated by Image J (Right). Data (A-B and E-F) represent the mean ± SD in different assays (n=3), *, P< 0.05; **, P< 0.01, determined by unpaired two-tailed t-test.
Figure Legend Snippet: miR-222-3p targets PDCD10 to repress cell migration by downregulating the Wnt/ β -catenin signaling pathway. (A) qPCR analysis of β -catenin and TCF4 expression in HO 8910 PM cells (PDCD10-overexpressing groups) and SKOV3 cells (PDCD10-silenced groups). ( B ) Activity of the Wnt/ β -catenin signaling pathway in HEK-293T cells detected by the TOP flash/FOP flash dual-luciferase reporting system. HEK-293T cells were transfected with TOP flash/FOP flash plasmid and pRL-SV40 vector followed by transfection with or without PDCD10, and siRNA-NC/PDCD10, siRNA-03/PDCD10, or siRNA-04. Firefly luciferase activities were tested by a luminometer, and the activity of the Wnt/ β -catenin signaling pathway was recorded as TOP/FOP. ( C ) Western blot analysis of PDCD10 and β -catenin protein expression following overexpression of PDCD10 in HO 8910 PM cell fractions. β -actin was utilized as a marker for the cytosolic fractions. ( D ) HO 8910 PM cells transduced with or without PDCD10 were subjected to immunofluorescence with human-specific β -catenin antibodies. ( β -catenin in red). Bar, 10 µm. ( E ) β -catenin nuclei-intensity (detected by Operetta High-Content Imaging System (Perkin Elmer) in HO 8910 PM cell). ( F ) Western blot analysis of PDCD10, β -catenin translation levels in HO 8910 PM cells, or after coculturing with miR-ctrl mimic or miR-222-3p mimic in the presence or absence of PDCD10 (Left). The relative expression rate was calculated by Image J (Right). Data (A-B and E-F) represent the mean ± SD in different assays (n=3), *, P< 0.05; **, P< 0.01, determined by unpaired two-tailed t-test.

Techniques Used: Migration, Expressing, Activity Assay, Luciferase, Transfection, Plasmid Preparation, Western Blot, Over Expression, Marker, Transduction, Immunofluorescence, Imaging, Two Tailed Test

SNAI2 increases cell migration via the SNAI2/miR-222-3p/PDCD10 axis and PDCD10-mediated promotion of EMT and Wnt/ β- catenin signaling. ( A ) Pearson's correlation scatter plots showed the fold changes of PDCD10 mRNA, miR-222-3p miRNA and SNAI2 mRNA levels in EOC tissues (n=38). ( B ) SNAI2 could enhance PDCD10 expression. PDCD10 mRNA expression levels were up-regulated after SNAI2 was overexpressed, but were down-regulated after SNAI2 was knocked down. ( C ) After transfection of SNAI2-overexpressing vectors and SNAI2 knockdown vectors (SNAI2 shRNA-01 and SNAI2 shRNA-02), the expression of PDCD10 was tested by Western blot in HO 8910 PM and SKOV3 cells. ( D ) Spearman correlation analysis of PDCD10, CDH1, VIM, SNAI2 and CTNNB1 levels in OC tissues (n=597) from the TCGA datasets. ( E ) A working model describing the interaction between SNAI2/miR-222-3p/PDCD10 during cancer metastasis. PDCD10 was critical for EOC cell mesenchymal movement and cell survival by maintaining low cell adhesion and high Wnt signaling. SNAI2 regulated PDCD10 by inhibiting pri-miR-222-3p expression and subsequent targeting of genes. SNAI2 regulated PDCD10 by inhibiting miR-222-3p expression, and further activated the downstream EMT signaling and Wnt/ β- catenin signaling to promote the expression of VIM, and β- catenin. These changes contributed to the EOC cell formation and migration, ultimately increasing tumor formation and metastasis. The data (B) represent the mean ± SD in different assays (n=3), **, P< 0.01; ***, P< 0.001, determined by unpaired two-tailed t-test.
Figure Legend Snippet: SNAI2 increases cell migration via the SNAI2/miR-222-3p/PDCD10 axis and PDCD10-mediated promotion of EMT and Wnt/ β- catenin signaling. ( A ) Pearson's correlation scatter plots showed the fold changes of PDCD10 mRNA, miR-222-3p miRNA and SNAI2 mRNA levels in EOC tissues (n=38). ( B ) SNAI2 could enhance PDCD10 expression. PDCD10 mRNA expression levels were up-regulated after SNAI2 was overexpressed, but were down-regulated after SNAI2 was knocked down. ( C ) After transfection of SNAI2-overexpressing vectors and SNAI2 knockdown vectors (SNAI2 shRNA-01 and SNAI2 shRNA-02), the expression of PDCD10 was tested by Western blot in HO 8910 PM and SKOV3 cells. ( D ) Spearman correlation analysis of PDCD10, CDH1, VIM, SNAI2 and CTNNB1 levels in OC tissues (n=597) from the TCGA datasets. ( E ) A working model describing the interaction between SNAI2/miR-222-3p/PDCD10 during cancer metastasis. PDCD10 was critical for EOC cell mesenchymal movement and cell survival by maintaining low cell adhesion and high Wnt signaling. SNAI2 regulated PDCD10 by inhibiting pri-miR-222-3p expression and subsequent targeting of genes. SNAI2 regulated PDCD10 by inhibiting miR-222-3p expression, and further activated the downstream EMT signaling and Wnt/ β- catenin signaling to promote the expression of VIM, and β- catenin. These changes contributed to the EOC cell formation and migration, ultimately increasing tumor formation and metastasis. The data (B) represent the mean ± SD in different assays (n=3), **, P< 0.01; ***, P< 0.001, determined by unpaired two-tailed t-test.

Techniques Used: Migration, Expressing, Transfection, shRNA, Western Blot, Two Tailed Test


Structured Review

Proteintech pdcd10
Expression of <t>PDCD10</t> in human pituitary adenomas. ( A ) A dataset (GSE26966) was obtained from the GEO database to compare the mRNA expression level of PDCD10 between PA tumor tissues (N=14) and normal pituitary tissue (N=9). ( B ) Relative mRNA expression levels of PDCD10 by RT-qPCR in invasive (n=15) and non-invasive (n=15) pituitary adenomas. Non-invasive values were set to 1. ( C ) Representative Western blots showed the expression level of PCDC10 protein (25 kDa) in invasive and noninvasive pituitary adenomas. Band intensities were quantified and normalized to GAPDH. ( D ) Representative images of immunohistochemistry evaluated the expression of PDCD10 in invasive and non-invasive (n=15) pituitary adenomas. **P < 0.01; ***P < 0.001; ****P < 0.0001.
Pdcd10, supplied by Proteintech, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/pdcd10/product/Proteintech
Average 93 stars, based on 1 article reviews
Price from $9.99 to $1999.99
pdcd10 - by Bioz Stars, 2024-07
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1) Product Images from "PDCD10 promotes the aggressive behaviors of pituitary adenomas by up-regulating CXCR2 and activating downstream AKT/ERK signaling"

Article Title: PDCD10 promotes the aggressive behaviors of pituitary adenomas by up-regulating CXCR2 and activating downstream AKT/ERK signaling

Journal: Aging (Albany NY)

doi: 10.18632/aging.204206

Expression of PDCD10 in human pituitary adenomas. ( A ) A dataset (GSE26966) was obtained from the GEO database to compare the mRNA expression level of PDCD10 between PA tumor tissues (N=14) and normal pituitary tissue (N=9). ( B ) Relative mRNA expression levels of PDCD10 by RT-qPCR in invasive (n=15) and non-invasive (n=15) pituitary adenomas. Non-invasive values were set to 1. ( C ) Representative Western blots showed the expression level of PCDC10 protein (25 kDa) in invasive and noninvasive pituitary adenomas. Band intensities were quantified and normalized to GAPDH. ( D ) Representative images of immunohistochemistry evaluated the expression of PDCD10 in invasive and non-invasive (n=15) pituitary adenomas. **P < 0.01; ***P < 0.001; ****P < 0.0001.
Figure Legend Snippet: Expression of PDCD10 in human pituitary adenomas. ( A ) A dataset (GSE26966) was obtained from the GEO database to compare the mRNA expression level of PDCD10 between PA tumor tissues (N=14) and normal pituitary tissue (N=9). ( B ) Relative mRNA expression levels of PDCD10 by RT-qPCR in invasive (n=15) and non-invasive (n=15) pituitary adenomas. Non-invasive values were set to 1. ( C ) Representative Western blots showed the expression level of PCDC10 protein (25 kDa) in invasive and noninvasive pituitary adenomas. Band intensities were quantified and normalized to GAPDH. ( D ) Representative images of immunohistochemistry evaluated the expression of PDCD10 in invasive and non-invasive (n=15) pituitary adenomas. **P < 0.01; ***P < 0.001; ****P < 0.0001.

Techniques Used: Expressing, Quantitative RT-PCR, Western Blot, Immunohistochemistry

PDCD10 silencing suppresses the proliferation, migration, invasion and EMT of PA cells. ( A ) Western blotting was performed to detect the impact of PDCD10 silencing on the expression of EMT markers in Att-20 cells and TtT/GF cells. ( B ) Band intensities were quantified and normalized to GAPDH. ( C , D ) CCK-8 assay was used to assess cell proliferation capacity after PDCD10 silencing in Att-20 and TtT/GF cells. ( E , F ) Scratch assay was used to examine the relative migration rates of Att-20 and TtT/GF cells (magnification:100x). ( G , H ) Transwell invasion assay was used to analyze the invasion potential of Att-20 and TtT/GF cells (magnification: 200x). * P < 0.05.
Figure Legend Snippet: PDCD10 silencing suppresses the proliferation, migration, invasion and EMT of PA cells. ( A ) Western blotting was performed to detect the impact of PDCD10 silencing on the expression of EMT markers in Att-20 cells and TtT/GF cells. ( B ) Band intensities were quantified and normalized to GAPDH. ( C , D ) CCK-8 assay was used to assess cell proliferation capacity after PDCD10 silencing in Att-20 and TtT/GF cells. ( E , F ) Scratch assay was used to examine the relative migration rates of Att-20 and TtT/GF cells (magnification:100x). ( G , H ) Transwell invasion assay was used to analyze the invasion potential of Att-20 and TtT/GF cells (magnification: 200x). * P < 0.05.

Techniques Used: Migration, Western Blot, Expressing, CCK-8 Assay, Wound Healing Assay, Transwell Invasion Assay

Overexpression of PDCD10 promotes the proliferation, migration, invasion and EMT of PA cells. ( A , B ) Western blotting was performed to detect the impact of PDCD10 overexpression on the expression levels of EMT markers in Att-20 cells and TtT/GF cells. Band intensities were quantified and normalized to GAPDH. ( C , D ) CCK-8 assay was used to assess cell proliferation potential after PDCD10 overexpression in Att-20 and TtT/GF cells. ( E , F ) Scratch assay was employed to examine the relative migration rates of Att-20 and TtT/GF cells after PDCD10 overexpression (magnification:100x). ( G , H ) Transwell invasion assay was used to detect the invasion potential of Att-20 and TtT/GF cells after PDCD10 overexpression (magnification: 200x). * P < 0.05.
Figure Legend Snippet: Overexpression of PDCD10 promotes the proliferation, migration, invasion and EMT of PA cells. ( A , B ) Western blotting was performed to detect the impact of PDCD10 overexpression on the expression levels of EMT markers in Att-20 cells and TtT/GF cells. Band intensities were quantified and normalized to GAPDH. ( C , D ) CCK-8 assay was used to assess cell proliferation potential after PDCD10 overexpression in Att-20 and TtT/GF cells. ( E , F ) Scratch assay was employed to examine the relative migration rates of Att-20 and TtT/GF cells after PDCD10 overexpression (magnification:100x). ( G , H ) Transwell invasion assay was used to detect the invasion potential of Att-20 and TtT/GF cells after PDCD10 overexpression (magnification: 200x). * P < 0.05.

Techniques Used: Over Expression, Migration, Western Blot, Expressing, CCK-8 Assay, Wound Healing Assay, Transwell Invasion Assay

PDCD10 alters the protein expression level of CXCR2 and regulates the activation of downstream AKT/ERK signal pathways. ( A , B ) Western blotting was used to detect the expression level of CXCR2 and phosphorylation level of AKT, ERK1/2 and STAT3 in Att-20 cells after PDCD10 silencing or overexpression. Band intensities were quantified and normalized to GAPDH. * P < 0.05. ( C , D ) In TtT/GF cells, western blotting was performed to examine the expression level of CXCR2 and phosphorylation level of AKT, ERK1/2 and STAT3 after PDCD10 silencing or overexpression. Band intensities were quantified and normalized to GAPDH. * P < 0.05.
Figure Legend Snippet: PDCD10 alters the protein expression level of CXCR2 and regulates the activation of downstream AKT/ERK signal pathways. ( A , B ) Western blotting was used to detect the expression level of CXCR2 and phosphorylation level of AKT, ERK1/2 and STAT3 in Att-20 cells after PDCD10 silencing or overexpression. Band intensities were quantified and normalized to GAPDH. * P < 0.05. ( C , D ) In TtT/GF cells, western blotting was performed to examine the expression level of CXCR2 and phosphorylation level of AKT, ERK1/2 and STAT3 after PDCD10 silencing or overexpression. Band intensities were quantified and normalized to GAPDH. * P < 0.05.

Techniques Used: Expressing, Activation Assay, Western Blot, Over Expression

Activation of CXCR2 rescues the inactivation of AKT/ERK signaling and the tumor-suppressive effects induced by PDCD10 silencing. ( A , B ) Western blotting was performed to examine the phosphorylation levels of AKT and ERK1/2 after recombinant CXCL2 administration (200ng/ml) in Att-20 and TtT/GF cells with PDCD10 silencing. ( C , D ) CCK-8 assay was used to evaluate cell proliferation potential after CXCL2 administration in Att-20 and TtT/GF cells with PDCD10 silencing. ( E , F ) Relative migration rate of ATT-20 and TtT/GF cells with PDCD10 silencing was analyzed by scratch assay after CXCL2 administration (magnification: 100x). ( G , H ) Transwell invasion assay was employed to assess the invasion capacity of Att-20 and TtT/GF cells with PDCD10 silencing after CXCL2 administration (magnification: 200x). * P < 0.05.
Figure Legend Snippet: Activation of CXCR2 rescues the inactivation of AKT/ERK signaling and the tumor-suppressive effects induced by PDCD10 silencing. ( A , B ) Western blotting was performed to examine the phosphorylation levels of AKT and ERK1/2 after recombinant CXCL2 administration (200ng/ml) in Att-20 and TtT/GF cells with PDCD10 silencing. ( C , D ) CCK-8 assay was used to evaluate cell proliferation potential after CXCL2 administration in Att-20 and TtT/GF cells with PDCD10 silencing. ( E , F ) Relative migration rate of ATT-20 and TtT/GF cells with PDCD10 silencing was analyzed by scratch assay after CXCL2 administration (magnification: 100x). ( G , H ) Transwell invasion assay was employed to assess the invasion capacity of Att-20 and TtT/GF cells with PDCD10 silencing after CXCL2 administration (magnification: 200x). * P < 0.05.

Techniques Used: Activation Assay, Western Blot, Recombinant, CCK-8 Assay, Migration, Wound Healing Assay, Transwell Invasion Assay

PDCD10 silencing impairs the tumorigenesis and reduces CXCR2 expression of PA cells in vivo . ( A , B ) Images for Att-20 and TtT/GF xenografts from nude mice (Left). Statistical analysis of xenograft tumor weights (Right). ( C , D ) Representative IHC images of Att-20 and TtT/GF xenograft tumor tissues for PDCD10, CXCR2 and Ki-67 staining (200x). Scale bars: 100μm. ( E , F ) Intensity of PDCD10, CXCR2 and Ki-67 staining were analyzed by IHC-Profiler. ( G , H ) Western blotting was performed to examine the expression of CXCR2 in Att-20 and TtT/GF xenograft tumor samples. Band intensities were quantified and normalized to GAPDH. * P < 0.05.
Figure Legend Snippet: PDCD10 silencing impairs the tumorigenesis and reduces CXCR2 expression of PA cells in vivo . ( A , B ) Images for Att-20 and TtT/GF xenografts from nude mice (Left). Statistical analysis of xenograft tumor weights (Right). ( C , D ) Representative IHC images of Att-20 and TtT/GF xenograft tumor tissues for PDCD10, CXCR2 and Ki-67 staining (200x). Scale bars: 100μm. ( E , F ) Intensity of PDCD10, CXCR2 and Ki-67 staining were analyzed by IHC-Profiler. ( G , H ) Western blotting was performed to examine the expression of CXCR2 in Att-20 and TtT/GF xenograft tumor samples. Band intensities were quantified and normalized to GAPDH. * P < 0.05.

Techniques Used: Expressing, In Vivo, Staining, Western Blot


Structured Review

Addgene inc human mrgprx2
Human Mrgprx2, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/human mrgprx2/product/Addgene inc
Average 93 stars, based on 1 article reviews
Price from $9.99 to $1999.99
human mrgprx2 - by Bioz Stars, 2024-07
93/100 stars

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Structured Review

Addgene inc mrgprx2 tango plasmid
Murepavadin induces intracellular Ca 2+ mobilization, degranulation and chemokine production in MCs through <t>MRGPRX2.</t> (A) Calcium mobilization measurement following murepavadin (10 μM) stimulation of Fura-2 loaded WT-RBL and RBL-MRGPRX2 cells. (B) WT-RBL and RBL-MRGPRX2 cells were exposed to 10 μM murepavadin (30 min), and degranulation was assayed by measuring the release of β-hexosaminidase. (C) RBL-MRGPRX2 cells were incubated in the presence or absence of pertussis toxin (PTx), at a concentration of 100 ng/mL for 16 h and exposed to indicated concentrations of murepavadin (30 min) and degranulation was assayed by measuring the release of β-hexosaminidase. (D, E) WT-RBL and RBL-MRGPRX2 cells were incubated in the presence or absence of pertussis toxin (PTx) at a concentration of 100 ng/mL for 16 h and exposed to 10 μM of murepavadin (24 h) and the production of cytokine TNF-α and chemokine CCL2 were quantified by ELISA. Data presented are the mean ± SEM of at least three experiments. Statistical significance was determined by two-way ANOVA with Šídák’s or Tukey’s multiple comparisons at a value * p < 0.05, *** p < 0.001, **** p < 0.0001, and ns denotes “not significant”.
Mrgprx2 Tango Plasmid, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Murepavadin, a Small Molecule Host Defense Peptide Mimetic, Activates Mast Cells via MRGPRX2 and MrgprB2"

Article Title: Murepavadin, a Small Molecule Host Defense Peptide Mimetic, Activates Mast Cells via MRGPRX2 and MrgprB2

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2021.689410

Murepavadin induces intracellular Ca 2+ mobilization, degranulation and chemokine production in MCs through MRGPRX2. (A) Calcium mobilization measurement following murepavadin (10 μM) stimulation of Fura-2 loaded WT-RBL and RBL-MRGPRX2 cells. (B) WT-RBL and RBL-MRGPRX2 cells were exposed to 10 μM murepavadin (30 min), and degranulation was assayed by measuring the release of β-hexosaminidase. (C) RBL-MRGPRX2 cells were incubated in the presence or absence of pertussis toxin (PTx), at a concentration of 100 ng/mL for 16 h and exposed to indicated concentrations of murepavadin (30 min) and degranulation was assayed by measuring the release of β-hexosaminidase. (D, E) WT-RBL and RBL-MRGPRX2 cells were incubated in the presence or absence of pertussis toxin (PTx) at a concentration of 100 ng/mL for 16 h and exposed to 10 μM of murepavadin (24 h) and the production of cytokine TNF-α and chemokine CCL2 were quantified by ELISA. Data presented are the mean ± SEM of at least three experiments. Statistical significance was determined by two-way ANOVA with Šídák’s or Tukey’s multiple comparisons at a value * p < 0.05, *** p < 0.001, **** p < 0.0001, and ns denotes “not significant”.
Figure Legend Snippet: Murepavadin induces intracellular Ca 2+ mobilization, degranulation and chemokine production in MCs through MRGPRX2. (A) Calcium mobilization measurement following murepavadin (10 μM) stimulation of Fura-2 loaded WT-RBL and RBL-MRGPRX2 cells. (B) WT-RBL and RBL-MRGPRX2 cells were exposed to 10 μM murepavadin (30 min), and degranulation was assayed by measuring the release of β-hexosaminidase. (C) RBL-MRGPRX2 cells were incubated in the presence or absence of pertussis toxin (PTx), at a concentration of 100 ng/mL for 16 h and exposed to indicated concentrations of murepavadin (30 min) and degranulation was assayed by measuring the release of β-hexosaminidase. (D, E) WT-RBL and RBL-MRGPRX2 cells were incubated in the presence or absence of pertussis toxin (PTx) at a concentration of 100 ng/mL for 16 h and exposed to 10 μM of murepavadin (24 h) and the production of cytokine TNF-α and chemokine CCL2 were quantified by ELISA. Data presented are the mean ± SEM of at least three experiments. Statistical significance was determined by two-way ANOVA with Šídák’s or Tukey’s multiple comparisons at a value * p < 0.05, *** p < 0.001, **** p < 0.0001, and ns denotes “not significant”.

Techniques Used: Incubation, Concentration Assay, Enzyme-linked Immunosorbent Assay

Naturally occurring MRGPRX2 missense variants are hypo-responsive to murepavadin for MC degranulation. (A) Snake diagram of MRGPRX2 indicating the two amino acid residues to be investigated. (B) Single amino acid substitution for each of the MRGPRX2 variant is shown in the table. (C) Cell surface receptor expression of the wild-type MRGPRX2 (WT) and its missense variants (G165E and V282M) was confirmed using flow cytometry. (D) RBL cells expressing MRGPRX2 (WT) and its variants (G165E and V282M) were exposed to 10 μM murepavadin (30 min) and degranulation was assayed by measuring the release of β-hexosaminidase. Statistical significance was determined by two-way ANOVA with Tukey’s multiple comparisons at a value ** p < 0.01 and **** p < 0.0001.
Figure Legend Snippet: Naturally occurring MRGPRX2 missense variants are hypo-responsive to murepavadin for MC degranulation. (A) Snake diagram of MRGPRX2 indicating the two amino acid residues to be investigated. (B) Single amino acid substitution for each of the MRGPRX2 variant is shown in the table. (C) Cell surface receptor expression of the wild-type MRGPRX2 (WT) and its missense variants (G165E and V282M) was confirmed using flow cytometry. (D) RBL cells expressing MRGPRX2 (WT) and its variants (G165E and V282M) were exposed to 10 μM murepavadin (30 min) and degranulation was assayed by measuring the release of β-hexosaminidase. Statistical significance was determined by two-way ANOVA with Tukey’s multiple comparisons at a value ** p < 0.01 and **** p < 0.0001.

Techniques Used: Variant Assay, Cell Surface Receptor Assay, Expressing, Flow Cytometry

Murepavadin does not promote β-arrestin recruitment following MRGPRX2 activation. (A, B) HTLA-MRGPRX2 cells were exposed to indicated concentrations of C48/80 or murepavadin for 16 h. Medium was removed and Bright-Glo solution (100 µL) was added into each well (96-well plate) and β-arrestin gene expression (in relative luminescence unit) was measured. (C, D) HTLA-MRGPRX2 cells were exposed to MRGPRX2 ligand (C48/80 or murepavadin, 16 h) and cell surface receptor expression of MRGPRX2 was confirmed using flow cytometry and quantified using a mean fluorescent intensity (MFI) in comparison to the untreated control. (E, F) Representative histograms of MRGPRX2 cell surface receptor expression of HTLA-MRGPRX2 cells. Statistical significance was determined by one-way ANOVA with Dunnett’s multiple comparisons at a value **** p < 0.0001 and ns denotes “not significant”.
Figure Legend Snippet: Murepavadin does not promote β-arrestin recruitment following MRGPRX2 activation. (A, B) HTLA-MRGPRX2 cells were exposed to indicated concentrations of C48/80 or murepavadin for 16 h. Medium was removed and Bright-Glo solution (100 µL) was added into each well (96-well plate) and β-arrestin gene expression (in relative luminescence unit) was measured. (C, D) HTLA-MRGPRX2 cells were exposed to MRGPRX2 ligand (C48/80 or murepavadin, 16 h) and cell surface receptor expression of MRGPRX2 was confirmed using flow cytometry and quantified using a mean fluorescent intensity (MFI) in comparison to the untreated control. (E, F) Representative histograms of MRGPRX2 cell surface receptor expression of HTLA-MRGPRX2 cells. Statistical significance was determined by one-way ANOVA with Dunnett’s multiple comparisons at a value **** p < 0.0001 and ns denotes “not significant”.

Techniques Used: Activation Assay, Expressing, Cell Surface Receptor Assay, Flow Cytometry

Murepavadin does not induce MRGPRX2 internalization or desensitization. (A) RBL-MRGPRX2 cells were exposed to MRGPRX2 ligand (C48/80 3 μg/mL or murepavadin 10 μM, 16 h) and cell surface receptor expression of MRGPRX2 was confirmed using flow cytometry. A representative histogram of MRGPRX2 cell surface receptor expression is shown. (B) Quantitative analysis of cell surface receptor expression was calculated using a mean fluorescent intensity (MFI) in comparison to the untreated control. (C, D) Calcium mobilization measurements of RBL-MRGPRX2 cells exposed to MRGPRX2 ligand (C48/80 3 μg/mL or murepavadin 10 μM, 16 h) following C48/80 (3 μg/mL) or murepavadin (10 μM) stimulation, respectively. Statistical significance was determined by one-way ANOVA with Dunnett’s multiple comparisons and two-way ANOVA with Šídák’s multiple comparisons at a value * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, and ns denotes “not significant”.
Figure Legend Snippet: Murepavadin does not induce MRGPRX2 internalization or desensitization. (A) RBL-MRGPRX2 cells were exposed to MRGPRX2 ligand (C48/80 3 μg/mL or murepavadin 10 μM, 16 h) and cell surface receptor expression of MRGPRX2 was confirmed using flow cytometry. A representative histogram of MRGPRX2 cell surface receptor expression is shown. (B) Quantitative analysis of cell surface receptor expression was calculated using a mean fluorescent intensity (MFI) in comparison to the untreated control. (C, D) Calcium mobilization measurements of RBL-MRGPRX2 cells exposed to MRGPRX2 ligand (C48/80 3 μg/mL or murepavadin 10 μM, 16 h) following C48/80 (3 μg/mL) or murepavadin (10 μM) stimulation, respectively. Statistical significance was determined by one-way ANOVA with Dunnett’s multiple comparisons and two-way ANOVA with Šídák’s multiple comparisons at a value * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, and ns denotes “not significant”.

Techniques Used: Cell Surface Receptor Assay, Expressing, Flow Cytometry

Proposed mechanism of action of murepavadin/MRGPRX2-induced bacterial clearance and wound healing. Murepavadin activates MCs via MRGPRX2. An increase in intracellular calcium following murepavadin stimulation triggers MC degranulation and chemokine production (IL-8, CCL3, CCL2, and TNF-α). These MC mediators induce immune cell recruitment and subsequently contribute to host defense and wound healing.
Figure Legend Snippet: Proposed mechanism of action of murepavadin/MRGPRX2-induced bacterial clearance and wound healing. Murepavadin activates MCs via MRGPRX2. An increase in intracellular calcium following murepavadin stimulation triggers MC degranulation and chemokine production (IL-8, CCL3, CCL2, and TNF-α). These MC mediators induce immune cell recruitment and subsequently contribute to host defense and wound healing.

Techniques Used:


Structured Review

Addgene inc mrgprx2 tango plasmid
Substance P (SP) is a balanced agonist for <t>MRGPRX2.</t> ( A ) HTLA cells stably expressing MRGPRX2 (HTLA-MRGPRX2) were exposed to different concentrations of SP for 16 h, and β-arrestin–mediated gene expression was determined. ( B ) Rat basophilic leukemia (RBL) cells stably expressing MRGPRX2 (RBL-MRGPRX2) were stimulated with different concentrations of SP for the times indicated, and level of receptor internalization was determined by flow cytometry. ( C ) RBL-MRGPRX2 cells were cultured in the absence (untreated) or presence of SP (30 μM) for 16 h. Cells were subsequently stimulated with different concentrations of SP for 30 min, and percent degranulation was determined by β-hexosaminidase release assay. All data points are the mean ± SEM of at least three experiments. For comparisons of two samples, two-tailed unpaired t -test was used. For comparisons of multiple samples to a control group, one–way analysis of variance (ANOVA) with Dunnett’s post-hoc test was used. *** p < 0.001 and **** p < 0.0001.
Mrgprx2 Tango Plasmid, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, 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
mrgprx2 tango plasmid - by Bioz Stars, 2024-07
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1) Product Images from "Substance P Serves as a Balanced Agonist for MRGPRX2 and a Single Tyrosine Residue Is Required for β-Arrestin Recruitment and Receptor Internalization"

Article Title: Substance P Serves as a Balanced Agonist for MRGPRX2 and a Single Tyrosine Residue Is Required for β-Arrestin Recruitment and Receptor Internalization

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms22105318

Substance P (SP) is a balanced agonist for MRGPRX2. ( A ) HTLA cells stably expressing MRGPRX2 (HTLA-MRGPRX2) were exposed to different concentrations of SP for 16 h, and β-arrestin–mediated gene expression was determined. ( B ) Rat basophilic leukemia (RBL) cells stably expressing MRGPRX2 (RBL-MRGPRX2) were stimulated with different concentrations of SP for the times indicated, and level of receptor internalization was determined by flow cytometry. ( C ) RBL-MRGPRX2 cells were cultured in the absence (untreated) or presence of SP (30 μM) for 16 h. Cells were subsequently stimulated with different concentrations of SP for 30 min, and percent degranulation was determined by β-hexosaminidase release assay. All data points are the mean ± SEM of at least three experiments. For comparisons of two samples, two-tailed unpaired t -test was used. For comparisons of multiple samples to a control group, one–way analysis of variance (ANOVA) with Dunnett’s post-hoc test was used. *** p < 0.001 and **** p < 0.0001.
Figure Legend Snippet: Substance P (SP) is a balanced agonist for MRGPRX2. ( A ) HTLA cells stably expressing MRGPRX2 (HTLA-MRGPRX2) were exposed to different concentrations of SP for 16 h, and β-arrestin–mediated gene expression was determined. ( B ) Rat basophilic leukemia (RBL) cells stably expressing MRGPRX2 (RBL-MRGPRX2) were stimulated with different concentrations of SP for the times indicated, and level of receptor internalization was determined by flow cytometry. ( C ) RBL-MRGPRX2 cells were cultured in the absence (untreated) or presence of SP (30 μM) for 16 h. Cells were subsequently stimulated with different concentrations of SP for 30 min, and percent degranulation was determined by β-hexosaminidase release assay. All data points are the mean ± SEM of at least three experiments. For comparisons of two samples, two-tailed unpaired t -test was used. For comparisons of multiple samples to a control group, one–way analysis of variance (ANOVA) with Dunnett’s post-hoc test was used. *** p < 0.001 and **** p < 0.0001.

Techniques Used: Stable Transfection, Expressing, Flow Cytometry, Cell Culture, Release Assay, Two Tailed Test

Pertussis toxin (PTx) inhibits SP-induced mast cell (MC) degranulation, but has no effect on β-arrestin recruitment and MRGPRX2 internalization. RBL-MRGPRX2 cells were cultured in the absence or presence of PTx (100 ng/mL, 16 h), and the effects of SP on ( A ) degranulation, ( B ) β-arrestin-mediated gene expression, and ( C ) MRGPRX2 internalization were determined. All data points are the mean ± SEM of at least three experiments. Statistical significance was determined by two-tailed unpaired t -test. **** p < 0.0001.
Figure Legend Snippet: Pertussis toxin (PTx) inhibits SP-induced mast cell (MC) degranulation, but has no effect on β-arrestin recruitment and MRGPRX2 internalization. RBL-MRGPRX2 cells were cultured in the absence or presence of PTx (100 ng/mL, 16 h), and the effects of SP on ( A ) degranulation, ( B ) β-arrestin-mediated gene expression, and ( C ) MRGPRX2 internalization were determined. All data points are the mean ± SEM of at least three experiments. Statistical significance was determined by two-tailed unpaired t -test. **** p < 0.0001.

Techniques Used: Cell Culture, Expressing, Two Tailed Test

Y279A mutation of MRGPRX2 abolishes β-arrestin recruitment in response to SP. ( A ) HTLA cells were transiently transfected with cDNA encoding either WT-MRGPRX2 or its Y279A mutant, and cell surface receptor expression was determined by flow cytometry using PE-anti-MRGPRX2 antibody. ( B ) Cells were stimulated with SP (30 μM) for 16 h, and β-arrestin–mediated gene expression was measured. All data points are the mean ± SEM of at least three experiments. Two-tailed unpaired t-test was used. ** p < 0.01. ( C ) RBL cells co-expressing WT MRGPRX2 or Y279A and βArr2-GFP were stimulated with SP (30 μM) for 1 min and β-arrestin translocation was investigated by confocal microscopy. Scale bar = 10 μm.
Figure Legend Snippet: Y279A mutation of MRGPRX2 abolishes β-arrestin recruitment in response to SP. ( A ) HTLA cells were transiently transfected with cDNA encoding either WT-MRGPRX2 or its Y279A mutant, and cell surface receptor expression was determined by flow cytometry using PE-anti-MRGPRX2 antibody. ( B ) Cells were stimulated with SP (30 μM) for 16 h, and β-arrestin–mediated gene expression was measured. All data points are the mean ± SEM of at least three experiments. Two-tailed unpaired t-test was used. ** p < 0.01. ( C ) RBL cells co-expressing WT MRGPRX2 or Y279A and βArr2-GFP were stimulated with SP (30 μM) for 1 min and β-arrestin translocation was investigated by confocal microscopy. Scale bar = 10 μm.

Techniques Used: Mutagenesis, Transfection, Cell Surface Receptor Assay, Expressing, Flow Cytometry, Two Tailed Test, Translocation Assay, Confocal Microscopy

Y279A mutation of MRGPRX2 impairs SP-induced receptor internalization. ( A ) HTLA transiently expressing WT-MRGPRX2 or its Y279A mutant were stimulated with SP (30 μM) for 30 min, and the receptor internalization was determined by flow cytometry. Representative histogram for cell surface receptor expression before (black line) and after SP stimulation (blue line) are shown. ( B ) The percentage of receptor internalization after SP stimulation was calculated. All data points are the mean ± SEM of at least three experiments. Two-tailed unpaired t -test was used. ** p < 0.001.
Figure Legend Snippet: Y279A mutation of MRGPRX2 impairs SP-induced receptor internalization. ( A ) HTLA transiently expressing WT-MRGPRX2 or its Y279A mutant were stimulated with SP (30 μM) for 30 min, and the receptor internalization was determined by flow cytometry. Representative histogram for cell surface receptor expression before (black line) and after SP stimulation (blue line) are shown. ( B ) The percentage of receptor internalization after SP stimulation was calculated. All data points are the mean ± SEM of at least three experiments. Two-tailed unpaired t -test was used. ** p < 0.001.

Techniques Used: Mutagenesis, Expressing, Flow Cytometry, Cell Surface Receptor Assay, Two Tailed Test

Y279A mutation of MRGPRX2 displays resistance to SP-induced receptor trafficking. ( A ) Schematic showing the dual-color labeling of cell surface and internalized receptors (modified from Carrodus et al., 2014 ). ( B ) Change in receptor trafficking was observed in RBL-2H3 cells expressing WT MRGPRX2 and Y279A mutant after SP stimulation (30 μM; 30 min). Scale bar = 10 μm.
Figure Legend Snippet: Y279A mutation of MRGPRX2 displays resistance to SP-induced receptor trafficking. ( A ) Schematic showing the dual-color labeling of cell surface and internalized receptors (modified from Carrodus et al., 2014 ). ( B ) Change in receptor trafficking was observed in RBL-2H3 cells expressing WT MRGPRX2 and Y279A mutant after SP stimulation (30 μM; 30 min). Scale bar = 10 μm.

Techniques Used: Mutagenesis, Labeling, Modification, Expressing


Structured Review

Addgene inc mrgprx2 tango
(A) LAD2 cells were stimulated with different concentrations of AG-30/5C (0.01 – 1.5 μM) for 30 min. Percent degranulation (β-hexosaminidase release) was determined. (B) LAD2 cells were stably transduced with scrambled control shRNA or lentivirus shRNA targeting human <t>MRGPRX2.</t> Western blotting was performed to determine MRGPRX2 expression. β-actin was used as a loading control. (C) Control and MRGPRX2 knockdown LAD2 cells were stimulated with AG-30/5C (1 μM) and degranulation was determine. (D) RBL-2H3 and RBL-2H3 cell stably expressing MRGPRX2 (RBL-MRGPRX2) were stimulated with different concentrations of AG-30/5C (0.01 – 1.5 μM) and percent degranulation was determined. All data are expressed as a mean ± SEM of three experiments. Statistical significance was determined by non-parametric t-Test and two way ANOVA (P value<.05). Western blotting data presented are representative of three similar experiments. *** indicates P value<.001,** indicates P value <.01 and * indicates P value <.05.
Mrgprx2 Tango, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 93 stars, based on 1 article reviews
Price from $9.99 to $1999.99
mrgprx2 tango - by Bioz Stars, 2024-07
93/100 stars

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1) Product Images from "Angiogenic Host Defense Peptide AG-30/5C and Bradykinin B 2 receptor antagonist Icatibant are G protein-biased agonists for MRGPRX2 in mast cells"

Article Title: Angiogenic Host Defense Peptide AG-30/5C and Bradykinin B 2 receptor antagonist Icatibant are G protein-biased agonists for MRGPRX2 in mast cells

Journal: Journal of immunology (Baltimore, Md. : 1950)

doi: 10.4049/jimmunol.1801227

(A) LAD2 cells were stimulated with different concentrations of AG-30/5C (0.01 – 1.5 μM) for 30 min. Percent degranulation (β-hexosaminidase release) was determined. (B) LAD2 cells were stably transduced with scrambled control shRNA or lentivirus shRNA targeting human MRGPRX2. Western blotting was performed to determine MRGPRX2 expression. β-actin was used as a loading control. (C) Control and MRGPRX2 knockdown LAD2 cells were stimulated with AG-30/5C (1 μM) and degranulation was determine. (D) RBL-2H3 and RBL-2H3 cell stably expressing MRGPRX2 (RBL-MRGPRX2) were stimulated with different concentrations of AG-30/5C (0.01 – 1.5 μM) and percent degranulation was determined. All data are expressed as a mean ± SEM of three experiments. Statistical significance was determined by non-parametric t-Test and two way ANOVA (P value<.05). Western blotting data presented are representative of three similar experiments. *** indicates P value<.001,** indicates P value <.01 and * indicates P value <.05.
Figure Legend Snippet: (A) LAD2 cells were stimulated with different concentrations of AG-30/5C (0.01 – 1.5 μM) for 30 min. Percent degranulation (β-hexosaminidase release) was determined. (B) LAD2 cells were stably transduced with scrambled control shRNA or lentivirus shRNA targeting human MRGPRX2. Western blotting was performed to determine MRGPRX2 expression. β-actin was used as a loading control. (C) Control and MRGPRX2 knockdown LAD2 cells were stimulated with AG-30/5C (1 μM) and degranulation was determine. (D) RBL-2H3 and RBL-2H3 cell stably expressing MRGPRX2 (RBL-MRGPRX2) were stimulated with different concentrations of AG-30/5C (0.01 – 1.5 μM) and percent degranulation was determined. All data are expressed as a mean ± SEM of three experiments. Statistical significance was determined by non-parametric t-Test and two way ANOVA (P value<.05). Western blotting data presented are representative of three similar experiments. *** indicates P value<.001,** indicates P value <.01 and * indicates P value <.05.

Techniques Used: Stable Transfection, Transduction, shRNA, Western Blot, Expressing

(A) RBL-MRGPRX2 cells cultured in absence (-PTx) or presence of pertussis toxin (+PTx, 100 ng/ml, 16h). Cells were stimulated with compound 48/80 (1μg/mL) or AG-30/5C (1 μM) and percent degranulation was determined. (B-C) RBL-MRGPRX2 cells were cultured in the absence or presence of PTx, loaded with Indo-1 (1 μM, 30 min) and stimulated with either compound 48/80 (1 μg/mL) or AG-30/5C (1 μM) as indicated by arrows and time course of calcium mobilization was determined. Representative traces of 3 similar experiments are shown. All data points for degranulation are expressed as mean ± SEM of three experiments. Statistical significance was determined by non-parametric t-Test and two way ANOVA (P value<.05). *** indicates P value<.001,** indicates P value <.01 and * indicates P value <.05.
Figure Legend Snippet: (A) RBL-MRGPRX2 cells cultured in absence (-PTx) or presence of pertussis toxin (+PTx, 100 ng/ml, 16h). Cells were stimulated with compound 48/80 (1μg/mL) or AG-30/5C (1 μM) and percent degranulation was determined. (B-C) RBL-MRGPRX2 cells were cultured in the absence or presence of PTx, loaded with Indo-1 (1 μM, 30 min) and stimulated with either compound 48/80 (1 μg/mL) or AG-30/5C (1 μM) as indicated by arrows and time course of calcium mobilization was determined. Representative traces of 3 similar experiments are shown. All data points for degranulation are expressed as mean ± SEM of three experiments. Statistical significance was determined by non-parametric t-Test and two way ANOVA (P value<.05). *** indicates P value<.001,** indicates P value <.01 and * indicates P value <.05.

Techniques Used: Cell Culture

(A) Modular design of Tango constructs. (B) Upon activation (1), β-arrestin is recruited to the C terminus of the receptor (2). This is followed by cleavage of the GPCR fusion protein at the TEV protease–cleavage site (3). Cleavage results in the release of the tTA transcription factor (4), which after transport to the nucleus activates transcription of the luciferase reporter gene (5). (C) Untransfected (HTLA) and stably transfected HTLA-MRGPRX2-Tango (HTLA-MRGPRX2) cells were incubated with anti-FLAG-PE antibody and cell surface receptor expression was determined by flow cytometry. A representative overlay histogram of MRGPRX2 expression is shown. (D) HTLA-MRGPRX2 cells were exposed to compound 48/80 (1.0 or 30 μg/mL) and AG-30/5C (1.0 and 10 μM) respectively and β-arrestin-mediated gene expression was determined. (E) Representative Ca2+ measurement traces for compound 48/80 (1 μg/mL and 30 μg/mL) and AG-30/5C (1 μM and 10 μM) are shown. All data points for the β-arrestin activation assay are expressed as a mean ± SEM of three experiments. Statistical significance was determined by non-parametric t-Test and two way ANOVA (P value<.05). *** indicates P value<.001,** indicates P value <.01 and * indicates P value <.05.
Figure Legend Snippet: (A) Modular design of Tango constructs. (B) Upon activation (1), β-arrestin is recruited to the C terminus of the receptor (2). This is followed by cleavage of the GPCR fusion protein at the TEV protease–cleavage site (3). Cleavage results in the release of the tTA transcription factor (4), which after transport to the nucleus activates transcription of the luciferase reporter gene (5). (C) Untransfected (HTLA) and stably transfected HTLA-MRGPRX2-Tango (HTLA-MRGPRX2) cells were incubated with anti-FLAG-PE antibody and cell surface receptor expression was determined by flow cytometry. A representative overlay histogram of MRGPRX2 expression is shown. (D) HTLA-MRGPRX2 cells were exposed to compound 48/80 (1.0 or 30 μg/mL) and AG-30/5C (1.0 and 10 μM) respectively and β-arrestin-mediated gene expression was determined. (E) Representative Ca2+ measurement traces for compound 48/80 (1 μg/mL and 30 μg/mL) and AG-30/5C (1 μM and 10 μM) are shown. All data points for the β-arrestin activation assay are expressed as a mean ± SEM of three experiments. Statistical significance was determined by non-parametric t-Test and two way ANOVA (P value<.05). *** indicates P value<.001,** indicates P value <.01 and * indicates P value <.05.

Techniques Used: Construct, Activation Assay, Luciferase, Stable Transfection, Transfection, Incubation, Cell Surface Receptor Assay, Expressing, Flow Cytometry

(A) RBL-MRGPRX2 cells (0.5 × 106) were cultured in the absence (unstimulated) or presence of compound 48/80 (1 μg/mL) or AG-30/5C (1 μM) for 16 h and cells surface expression of MRGPRX2 was determined by flow cytometry using anti-MRGPRX2-PE antibody. The data is presented as mean fluorescent intensity (MFI) of three experiments (B) A representative histogram of cell surface MRGPRX2 receptor expression is shown. (C) LAD2 cells were cultured in the absence (Control) or presence of in compound 48/80 (1 μg/mL) or AG-30/5C (1 μM) for 16h, washed and plated in a 96 well plate (10,000 cells/well). Cells were stimulated with compound 48/80 (1.0 μg/mL) and AG-30/5C (1 μM) respectively for 30 min and percent degranulation was determined by β-hexosaminidase release assay. All the points are expressed as a mean ± SEM of three experiments. Statistical significance was determined by non-parametric t-Test and two way ANOVA. *** indicates P value<.001,** indicates P value <.01 and * indicates P value <.05.
Figure Legend Snippet: (A) RBL-MRGPRX2 cells (0.5 × 106) were cultured in the absence (unstimulated) or presence of compound 48/80 (1 μg/mL) or AG-30/5C (1 μM) for 16 h and cells surface expression of MRGPRX2 was determined by flow cytometry using anti-MRGPRX2-PE antibody. The data is presented as mean fluorescent intensity (MFI) of three experiments (B) A representative histogram of cell surface MRGPRX2 receptor expression is shown. (C) LAD2 cells were cultured in the absence (Control) or presence of in compound 48/80 (1 μg/mL) or AG-30/5C (1 μM) for 16h, washed and plated in a 96 well plate (10,000 cells/well). Cells were stimulated with compound 48/80 (1.0 μg/mL) and AG-30/5C (1 μM) respectively for 30 min and percent degranulation was determined by β-hexosaminidase release assay. All the points are expressed as a mean ± SEM of three experiments. Statistical significance was determined by non-parametric t-Test and two way ANOVA. *** indicates P value<.001,** indicates P value <.01 and * indicates P value <.05.

Techniques Used: Cell Culture, Expressing, Flow Cytometry, Release Assay

(A) HTLA cells stably expressing MRGPRX2-Tango were incubated with resveratrol (100 μM, 5 min) followed by overnight stimulation with compound 48/80 (3.0, 10 and 20 μg/mL) or AG-30/5C (10 μM) respectively and relative luminescence was determined. (B) RBL-MRGPRX2 cells and (C) LAD2 cells either left untreated (Control) or incubated with resveratrol (100 μM, 5 min) and stimulated with compound 48/80 (1μg/mL) or AG-30/5C (1 μM) for 30 min and percent degranulation was determined. Statistical significance was determined by non-parametric t-Test and two way ANOVA. *** indicates P value<.001,** indicates P value <.01 and * indicates P value <.05.
Figure Legend Snippet: (A) HTLA cells stably expressing MRGPRX2-Tango were incubated with resveratrol (100 μM, 5 min) followed by overnight stimulation with compound 48/80 (3.0, 10 and 20 μg/mL) or AG-30/5C (10 μM) respectively and relative luminescence was determined. (B) RBL-MRGPRX2 cells and (C) LAD2 cells either left untreated (Control) or incubated with resveratrol (100 μM, 5 min) and stimulated with compound 48/80 (1μg/mL) or AG-30/5C (1 μM) for 30 min and percent degranulation was determined. Statistical significance was determined by non-parametric t-Test and two way ANOVA. *** indicates P value<.001,** indicates P value <.01 and * indicates P value <.05.

Techniques Used: Stable Transfection, Expressing, Incubation

(A and B) RBL-MRGPRX2 cells were cultured in the absence or presence of PTx (100 ng/mL, 16 h) and percent degranulation and Ca2+ mobilization in response to Icatibant (20 μg/mL) was determined as described for compound 48/80 and AG-30/5C (Figure 2). (C) RBL-MRGPRX2 and (D) LAD2 cells were incubated with resveratrol (100 μM, 5 min), stimulated with Icatibant (20 μg/mL) for 30 min and percent degranulation was determined. (E) HTLA cells expressing MRGPRX2 (HTLA-MRGPRX2) were exposed to medium (Control) or resveratrol (100 μM of 5 min) followed by overnight stimulation with compound 48/80 (10 μg/mL) or Icatibant (30 μg/mL) and chemiluminescence was measured (F) Representative Ca2+ traces for compound 48/80 (30 μg/mL) and Icatibant (30 μg/mL) in HTLA-MRGPRX2 cells are shown. Statistical significance was determined by non-parametric t-Test and two way ANOVA. *** indicates P value<.001,** indicates P value <.01 and * indicates P value <.05.
Figure Legend Snippet: (A and B) RBL-MRGPRX2 cells were cultured in the absence or presence of PTx (100 ng/mL, 16 h) and percent degranulation and Ca2+ mobilization in response to Icatibant (20 μg/mL) was determined as described for compound 48/80 and AG-30/5C (Figure 2). (C) RBL-MRGPRX2 and (D) LAD2 cells were incubated with resveratrol (100 μM, 5 min), stimulated with Icatibant (20 μg/mL) for 30 min and percent degranulation was determined. (E) HTLA cells expressing MRGPRX2 (HTLA-MRGPRX2) were exposed to medium (Control) or resveratrol (100 μM of 5 min) followed by overnight stimulation with compound 48/80 (10 μg/mL) or Icatibant (30 μg/mL) and chemiluminescence was measured (F) Representative Ca2+ traces for compound 48/80 (30 μg/mL) and Icatibant (30 μg/mL) in HTLA-MRGPRX2 cells are shown. Statistical significance was determined by non-parametric t-Test and two way ANOVA. *** indicates P value<.001,** indicates P value <.01 and * indicates P value <.05.

Techniques Used: Cell Culture, Incubation, Expressing

We propose that in addition to its direct antimicrobial activity, AG-30/5C contributed to host defense via MC degranulation through MRGPRX2. AG-30/5C contributes to wound healing via its action on vascular endothelial cells keratinocyte proliferation and migration through MRGPRX3 and MRGPRX4.
Figure Legend Snippet: We propose that in addition to its direct antimicrobial activity, AG-30/5C contributed to host defense via MC degranulation through MRGPRX2. AG-30/5C contributes to wound healing via its action on vascular endothelial cells keratinocyte proliferation and migration through MRGPRX3 and MRGPRX4.

Techniques Used: Activity Assay, Migration

rabbit polyclonal anti gluk2 antibody 66440  (Abcam)

 
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    Abcam rabbit polyclonal anti gluk2 antibody 66440
    Rabbit Polyclonal Anti Gluk2 Antibody 66440, supplied by Abcam, 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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    Addgene inc vector mrgprx2 tango
    (A) Effects on cells stably expressing myc-B 2 Rs from either species (positive control, BK). (B) Effect on cells transiently expressing human <t>MRGPRX2</t> (positive control, compound 48/80). Tracings, obtained via Fura-2 fluorescence, represent calcium mobilization, in fold of basal values to show the time course of effects elicited by receptor ligands (conventionally applied at time 15 s, arrows). The number of determinations is indicated between parentheses.
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    1) Product Images from "Species-specific pharmacology of maximakinin, an amphibian homologue of bradykinin: putative prodrug activity at the human B 2 receptor and peptidase resistance in rats"

    Article Title: Species-specific pharmacology of maximakinin, an amphibian homologue of bradykinin: putative prodrug activity at the human B 2 receptor and peptidase resistance in rats

    Journal: PeerJ

    doi: 10.7717/peerj.2911

    (A) Effects on cells stably expressing myc-B 2 Rs from either species (positive control, BK). (B) Effect on cells transiently expressing human MRGPRX2 (positive control, compound 48/80). Tracings, obtained via Fura-2 fluorescence, represent calcium mobilization, in fold of basal values to show the time course of effects elicited by receptor ligands (conventionally applied at time 15 s, arrows). The number of determinations is indicated between parentheses.
    Figure Legend Snippet: (A) Effects on cells stably expressing myc-B 2 Rs from either species (positive control, BK). (B) Effect on cells transiently expressing human MRGPRX2 (positive control, compound 48/80). Tracings, obtained via Fura-2 fluorescence, represent calcium mobilization, in fold of basal values to show the time course of effects elicited by receptor ligands (conventionally applied at time 15 s, arrows). The number of determinations is indicated between parentheses.

    Techniques Used: Stable Transfection, Expressing, Positive Control, Fluorescence


    Structured Review

    Proteintech rabbit polyclonal antibody against pdcd10
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    Rabbit Polyclonal Antibody Against Pdcd10, supplied by Proteintech, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Mutations in 2 distinct genetic pathways result in cerebral cavernous malformations in mice"

    Article Title: Mutations in 2 distinct genetic pathways result in cerebral cavernous malformations in mice

    Journal: The Journal of Clinical Investigation

    doi: 10.1172/JCI44393

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    Proteintech rabbit polyclonal antibody against pdcd10
    Table 1
    Rabbit Polyclonal Antibody Against Pdcd10, supplied by Proteintech, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Mutations in 2 distinct genetic pathways result in cerebral cavernous malformations in mice"

    Article Title: Mutations in 2 distinct genetic pathways result in cerebral cavernous malformations in mice

    Journal: The Journal of Clinical Investigation

    doi: 10.1172/JCI44393

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    Proteintech antibodies against pdcd10
    miR-222-3p directly suppresses <t>PDCD10</t> expression by binding to its 3' -UTR and inhibits EOC cell migration in vitro . ( A ) A Venn diagram was used to look for candidate genes targeted by miR-222-3p. ( B ) Levels of 15 candidate genes in HO 8910 PM cells transfected with miR-222-3p mimic. ( C ) miR-222-3p inhibitor assay in SKOV3 cells showing four candidate genes with upregulation of two genes, the most significant being PDCD10. ( D ) Western blot analysis of PDCD10 levels in two different EOC cell lines after transfection with miR-222-3p mimic or inhibitor. ( E ) Schematic depiction of the double-strand formation by miR-222-3p with the 3' -UTR of PDCD10. ( F ) Relative luciferase activities in HEK-293T cells co-transfected with a miR-222-3p mimic/inhibitor and PDCD10 WT/MUT. “++” stands for the double concentration. ( G and H ) Transwell and wound healing assays revealed inhibition of migration when HO 8910 PM cells were transfected with miR-222-3p mimic. Recovery assays showed that miR-222-3p suppressed migration of EOC cell lines due to its inhibitory effect on PDCD10 (Left). Cell counting and wound healing were quantified (Right). ( I ) qPCR and ( J ) Western blot analyses of PDCD10 expression levels in HO 8910 PM cells, and Image J calculated the relative expression rate. Data are means ± SEM. Data are from six (B) experiments and representative of three (C and F-J) independent experiments. *, P< 0.05; **, P< 0.01; ***, P< 0.001; ****, P< 0.0001, determined by unpaired two-tailed t-test.
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    Proteintech pdcd10
    Expression of <t>PDCD10</t> in human pituitary adenomas. ( A ) A dataset (GSE26966) was obtained from the GEO database to compare the mRNA expression level of PDCD10 between PA tumor tissues (N=14) and normal pituitary tissue (N=9). ( B ) Relative mRNA expression levels of PDCD10 by RT-qPCR in invasive (n=15) and non-invasive (n=15) pituitary adenomas. Non-invasive values were set to 1. ( C ) Representative Western blots showed the expression level of PCDC10 protein (25 kDa) in invasive and noninvasive pituitary adenomas. Band intensities were quantified and normalized to GAPDH. ( D ) Representative images of immunohistochemistry evaluated the expression of PDCD10 in invasive and non-invasive (n=15) pituitary adenomas. **P < 0.01; ***P < 0.001; ****P < 0.0001.
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    Addgene inc human mrgprx2
    Expression of <t>PDCD10</t> in human pituitary adenomas. ( A ) A dataset (GSE26966) was obtained from the GEO database to compare the mRNA expression level of PDCD10 between PA tumor tissues (N=14) and normal pituitary tissue (N=9). ( B ) Relative mRNA expression levels of PDCD10 by RT-qPCR in invasive (n=15) and non-invasive (n=15) pituitary adenomas. Non-invasive values were set to 1. ( C ) Representative Western blots showed the expression level of PCDC10 protein (25 kDa) in invasive and noninvasive pituitary adenomas. Band intensities were quantified and normalized to GAPDH. ( D ) Representative images of immunohistochemistry evaluated the expression of PDCD10 in invasive and non-invasive (n=15) pituitary adenomas. **P < 0.01; ***P < 0.001; ****P < 0.0001.
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    Addgene inc mrgprx2 tango plasmid
    Murepavadin induces intracellular Ca 2+ mobilization, degranulation and chemokine production in MCs through <t>MRGPRX2.</t> (A) Calcium mobilization measurement following murepavadin (10 μM) stimulation of Fura-2 loaded WT-RBL and RBL-MRGPRX2 cells. (B) WT-RBL and RBL-MRGPRX2 cells were exposed to 10 μM murepavadin (30 min), and degranulation was assayed by measuring the release of β-hexosaminidase. (C) RBL-MRGPRX2 cells were incubated in the presence or absence of pertussis toxin (PTx), at a concentration of 100 ng/mL for 16 h and exposed to indicated concentrations of murepavadin (30 min) and degranulation was assayed by measuring the release of β-hexosaminidase. (D, E) WT-RBL and RBL-MRGPRX2 cells were incubated in the presence or absence of pertussis toxin (PTx) at a concentration of 100 ng/mL for 16 h and exposed to 10 μM of murepavadin (24 h) and the production of cytokine TNF-α and chemokine CCL2 were quantified by ELISA. Data presented are the mean ± SEM of at least three experiments. Statistical significance was determined by two-way ANOVA with Šídák’s or Tukey’s multiple comparisons at a value * p < 0.05, *** p < 0.001, **** p < 0.0001, and ns denotes “not significant”.
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    (A) LAD2 cells were stimulated with different concentrations of AG-30/5C (0.01 – 1.5 μM) for 30 min. Percent degranulation (β-hexosaminidase release) was determined. (B) LAD2 cells were stably transduced with scrambled control shRNA or lentivirus shRNA targeting human <t>MRGPRX2.</t> Western blotting was performed to determine MRGPRX2 expression. β-actin was used as a loading control. (C) Control and MRGPRX2 knockdown LAD2 cells were stimulated with AG-30/5C (1 μM) and degranulation was determine. (D) RBL-2H3 and RBL-2H3 cell stably expressing MRGPRX2 (RBL-MRGPRX2) were stimulated with different concentrations of AG-30/5C (0.01 – 1.5 μM) and percent degranulation was determined. All data are expressed as a mean ± SEM of three experiments. Statistical significance was determined by non-parametric t-Test and two way ANOVA (P value<.05). Western blotting data presented are representative of three similar experiments. *** indicates P value<.001,** indicates P value <.01 and * indicates P value <.05.
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    Abcam rabbit polyclonal anti gluk2 antibody 66440
    (A) LAD2 cells were stimulated with different concentrations of AG-30/5C (0.01 – 1.5 μM) for 30 min. Percent degranulation (β-hexosaminidase release) was determined. (B) LAD2 cells were stably transduced with scrambled control shRNA or lentivirus shRNA targeting human <t>MRGPRX2.</t> Western blotting was performed to determine MRGPRX2 expression. β-actin was used as a loading control. (C) Control and MRGPRX2 knockdown LAD2 cells were stimulated with AG-30/5C (1 μM) and degranulation was determine. (D) RBL-2H3 and RBL-2H3 cell stably expressing MRGPRX2 (RBL-MRGPRX2) were stimulated with different concentrations of AG-30/5C (0.01 – 1.5 μM) and percent degranulation was determined. All data are expressed as a mean ± SEM of three experiments. Statistical significance was determined by non-parametric t-Test and two way ANOVA (P value<.05). Western blotting data presented are representative of three similar experiments. *** indicates P value<.001,** indicates P value <.01 and * indicates P value <.05.
    Rabbit Polyclonal Anti Gluk2 Antibody 66440, supplied by Abcam, 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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    Addgene inc vector mrgprx2 tango
    (A) Effects on cells stably expressing myc-B 2 Rs from either species (positive control, BK). (B) Effect on cells transiently expressing human <t>MRGPRX2</t> (positive control, compound 48/80). Tracings, obtained via Fura-2 fluorescence, represent calcium mobilization, in fold of basal values to show the time course of effects elicited by receptor ligands (conventionally applied at time 15 s, arrows). The number of determinations is indicated between parentheses.
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    Proteintech rabbit polyclonal antibody against pdcd10
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    Rabbit Polyclonal Antibody Against Pdcd10, supplied by Proteintech, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    miR-222-3p directly suppresses PDCD10 expression by binding to its 3' -UTR and inhibits EOC cell migration in vitro . ( A ) A Venn diagram was used to look for candidate genes targeted by miR-222-3p. ( B ) Levels of 15 candidate genes in HO 8910 PM cells transfected with miR-222-3p mimic. ( C ) miR-222-3p inhibitor assay in SKOV3 cells showing four candidate genes with upregulation of two genes, the most significant being PDCD10. ( D ) Western blot analysis of PDCD10 levels in two different EOC cell lines after transfection with miR-222-3p mimic or inhibitor. ( E ) Schematic depiction of the double-strand formation by miR-222-3p with the 3' -UTR of PDCD10. ( F ) Relative luciferase activities in HEK-293T cells co-transfected with a miR-222-3p mimic/inhibitor and PDCD10 WT/MUT. “++” stands for the double concentration. ( G and H ) Transwell and wound healing assays revealed inhibition of migration when HO 8910 PM cells were transfected with miR-222-3p mimic. Recovery assays showed that miR-222-3p suppressed migration of EOC cell lines due to its inhibitory effect on PDCD10 (Left). Cell counting and wound healing were quantified (Right). ( I ) qPCR and ( J ) Western blot analyses of PDCD10 expression levels in HO 8910 PM cells, and Image J calculated the relative expression rate. Data are means ± SEM. Data are from six (B) experiments and representative of three (C and F-J) independent experiments. *, P< 0.05; **, P< 0.01; ***, P< 0.001; ****, P< 0.0001, determined by unpaired two-tailed t-test.

    Journal: Theranostics

    Article Title: Non-canonical signaling pathway of SNAI2 induces EMT in ovarian cancer cells by suppressing miR-222-3p transcription and upregulating PDCD10

    doi: 10.7150/thno.43198

    Figure Lengend Snippet: miR-222-3p directly suppresses PDCD10 expression by binding to its 3' -UTR and inhibits EOC cell migration in vitro . ( A ) A Venn diagram was used to look for candidate genes targeted by miR-222-3p. ( B ) Levels of 15 candidate genes in HO 8910 PM cells transfected with miR-222-3p mimic. ( C ) miR-222-3p inhibitor assay in SKOV3 cells showing four candidate genes with upregulation of two genes, the most significant being PDCD10. ( D ) Western blot analysis of PDCD10 levels in two different EOC cell lines after transfection with miR-222-3p mimic or inhibitor. ( E ) Schematic depiction of the double-strand formation by miR-222-3p with the 3' -UTR of PDCD10. ( F ) Relative luciferase activities in HEK-293T cells co-transfected with a miR-222-3p mimic/inhibitor and PDCD10 WT/MUT. “++” stands for the double concentration. ( G and H ) Transwell and wound healing assays revealed inhibition of migration when HO 8910 PM cells were transfected with miR-222-3p mimic. Recovery assays showed that miR-222-3p suppressed migration of EOC cell lines due to its inhibitory effect on PDCD10 (Left). Cell counting and wound healing were quantified (Right). ( I ) qPCR and ( J ) Western blot analyses of PDCD10 expression levels in HO 8910 PM cells, and Image J calculated the relative expression rate. Data are means ± SEM. Data are from six (B) experiments and representative of three (C and F-J) independent experiments. *, P< 0.05; **, P< 0.01; ***, P< 0.001; ****, P< 0.0001, determined by unpaired two-tailed t-test.

    Article Snippet: PDCD10, SNAI2, E-cad, VIM and β- catenin used in this study were from following sources: antibodies against PDCD10 and GAPDH were purchased from Abcam and Utibody, respectively (ab110531 and UM4002, respectively); antibodies against SNAI2 and GAPDH were acquired from Affinity (AF4002) and Utibody (UM4001), respectively; antibodies against E-cad, VIM, and β- catenin (20874-1-AP, 22018-1-AP and 10366-1-AP, respectively) were obtained from Proteintech.

    Techniques: Expressing, Binding Assay, Migration, In Vitro, Transfection, Western Blot, Luciferase, Concentration Assay, Inhibition, Cell Counting, Two Tailed Test

    miR-222-3p suppresses EOC tumor metastasis in vivo by targeting PDCD10. ( A ) Schematic presentation of in vivo adhesion for equivalent numbers of GFP-labeled LV-miR-ctrl/LV-miR-222-3p and GFP-labeled ctrl vector/PDCD10 transfected stably in HO 8910 PM cells. Bar, 100 µm. ( B and C ) Representative images and quantification of intraperitoneal metastases in mice implanted intraperitoneally with the same number of HO 8910 PM cells (n= 4 mice per group). Bar, 1 cm. ( D ) Western blot analysis of PDCD10 levels in representative EOC metastatic nodules. ( E ) Representative images and bioluminescence mice images of lung tissue metastatic nodules 5 weeks (wk) after implantation. Bar, 0.5 cm. ( F ) Quantification of metastatic nodules in lung tissues of mice. ( G ) Representative images (Down) and metastatic nodule plots (Up) of mice stomach tissues formed in the restoration group. Bar, 0.5 cm. ( H ) IHC staining for PDCD10 in the stomach tissues of mice 5 weeks after implantation. Bar, 100 µm. ( I ) Representative images (Down) and metastatic nodule plots (Up) of mice liver tissues formed in the restoration group. Bar, 1 cm. ( J ) Representative HE staining of the mice lung tissues obtained from 5 weeks after implantation. Bar, 50 µm (Left) and 100 µm (Right). Data are means ± SEM and are representative of three (C, F, G, I) independent experiments. *, P< 0.05. **; P< 0.01; ***, P< 0.001, determined by unpaired two-tailed t-test.

    Journal: Theranostics

    Article Title: Non-canonical signaling pathway of SNAI2 induces EMT in ovarian cancer cells by suppressing miR-222-3p transcription and upregulating PDCD10

    doi: 10.7150/thno.43198

    Figure Lengend Snippet: miR-222-3p suppresses EOC tumor metastasis in vivo by targeting PDCD10. ( A ) Schematic presentation of in vivo adhesion for equivalent numbers of GFP-labeled LV-miR-ctrl/LV-miR-222-3p and GFP-labeled ctrl vector/PDCD10 transfected stably in HO 8910 PM cells. Bar, 100 µm. ( B and C ) Representative images and quantification of intraperitoneal metastases in mice implanted intraperitoneally with the same number of HO 8910 PM cells (n= 4 mice per group). Bar, 1 cm. ( D ) Western blot analysis of PDCD10 levels in representative EOC metastatic nodules. ( E ) Representative images and bioluminescence mice images of lung tissue metastatic nodules 5 weeks (wk) after implantation. Bar, 0.5 cm. ( F ) Quantification of metastatic nodules in lung tissues of mice. ( G ) Representative images (Down) and metastatic nodule plots (Up) of mice stomach tissues formed in the restoration group. Bar, 0.5 cm. ( H ) IHC staining for PDCD10 in the stomach tissues of mice 5 weeks after implantation. Bar, 100 µm. ( I ) Representative images (Down) and metastatic nodule plots (Up) of mice liver tissues formed in the restoration group. Bar, 1 cm. ( J ) Representative HE staining of the mice lung tissues obtained from 5 weeks after implantation. Bar, 50 µm (Left) and 100 µm (Right). Data are means ± SEM and are representative of three (C, F, G, I) independent experiments. *, P< 0.05. **; P< 0.01; ***, P< 0.001, determined by unpaired two-tailed t-test.

    Article Snippet: PDCD10, SNAI2, E-cad, VIM and β- catenin used in this study were from following sources: antibodies against PDCD10 and GAPDH were purchased from Abcam and Utibody, respectively (ab110531 and UM4002, respectively); antibodies against SNAI2 and GAPDH were acquired from Affinity (AF4002) and Utibody (UM4001), respectively; antibodies against E-cad, VIM, and β- catenin (20874-1-AP, 22018-1-AP and 10366-1-AP, respectively) were obtained from Proteintech.

    Techniques: In Vivo, Labeling, Plasmid Preparation, Transfection, Stable Transfection, Western Blot, Immunohistochemistry, Staining, Two Tailed Test

    miR-222-3p targets PDCD10 to inhibit cell migration via EMT pathway. ( A ) Meta-analysis describing forest plots of PDCD10 expression as a univariate predictor of overall survival. ( B ) Kaplan-Meier curves for overall survival probability in 1656 OC patients with low (n=640) and high (n=1016) PDCD10 expression (analyzed with log-rank test) from KMplot ( http://kmplot.com/analysis/ ). ( C ) qPCR and Western blot analyses of PDCD10 levels in FE25 and 4 different EOC cell lines. ( D ) Representative images of PDCD10 expression in normal and tumor ovary tissues. Bar, 100 µm (Left) and 30 µm (Right). ( E ) Enrichment evaluation within the PDCD10-expressing levels for predicted GSEA results of TCGA reference gene sets for high and low PDCD10 expression groups. Genes expressed in the profile datasets were ranked by fold changes (high-expression/low-expression). GSEA correlation pathways were determined by the given algorithm. Vertical bars along the x-axis of the GSEA diagram represent the positions within the ranked list of genes set in the given sets. Positive and negative GSEA curves mean positive and negative enrichments, respectively. ( F and G ) qPCR and Western blot analyses of PDCD10, E-cad (CDH1) and VIM expression in HO 8910 PM cells (PDCD10-overexpressing groups) and SKOV3 cells (PDCD10-silenced groups). ( H ) HO 8910 PM cells transduced with control vector or PDCD10 (GFP-labeled ctrl vector and PDCD10 all in green) were subjected to immunofluorescence with human-specific E-cad and VIM antibodies (in red). Bar, 10 µm. ( I ) Western blot analysis of PDCD10, E-cad, Vim translation levels in HO 8910 PM cells after treatment with miR-ctrl mimic or miR-222-3p mimic, in the presence or absence of PDCD10. Data are means ± SEM and are representative of three (C and F) independent experiments. *, P< 0.05; **, P< 0.01; ***, P< 0.001; ****, P< 0.0001, determined by unpaired two-tailed t-test.

    Journal: Theranostics

    Article Title: Non-canonical signaling pathway of SNAI2 induces EMT in ovarian cancer cells by suppressing miR-222-3p transcription and upregulating PDCD10

    doi: 10.7150/thno.43198

    Figure Lengend Snippet: miR-222-3p targets PDCD10 to inhibit cell migration via EMT pathway. ( A ) Meta-analysis describing forest plots of PDCD10 expression as a univariate predictor of overall survival. ( B ) Kaplan-Meier curves for overall survival probability in 1656 OC patients with low (n=640) and high (n=1016) PDCD10 expression (analyzed with log-rank test) from KMplot ( http://kmplot.com/analysis/ ). ( C ) qPCR and Western blot analyses of PDCD10 levels in FE25 and 4 different EOC cell lines. ( D ) Representative images of PDCD10 expression in normal and tumor ovary tissues. Bar, 100 µm (Left) and 30 µm (Right). ( E ) Enrichment evaluation within the PDCD10-expressing levels for predicted GSEA results of TCGA reference gene sets for high and low PDCD10 expression groups. Genes expressed in the profile datasets were ranked by fold changes (high-expression/low-expression). GSEA correlation pathways were determined by the given algorithm. Vertical bars along the x-axis of the GSEA diagram represent the positions within the ranked list of genes set in the given sets. Positive and negative GSEA curves mean positive and negative enrichments, respectively. ( F and G ) qPCR and Western blot analyses of PDCD10, E-cad (CDH1) and VIM expression in HO 8910 PM cells (PDCD10-overexpressing groups) and SKOV3 cells (PDCD10-silenced groups). ( H ) HO 8910 PM cells transduced with control vector or PDCD10 (GFP-labeled ctrl vector and PDCD10 all in green) were subjected to immunofluorescence with human-specific E-cad and VIM antibodies (in red). Bar, 10 µm. ( I ) Western blot analysis of PDCD10, E-cad, Vim translation levels in HO 8910 PM cells after treatment with miR-ctrl mimic or miR-222-3p mimic, in the presence or absence of PDCD10. Data are means ± SEM and are representative of three (C and F) independent experiments. *, P< 0.05; **, P< 0.01; ***, P< 0.001; ****, P< 0.0001, determined by unpaired two-tailed t-test.

    Article Snippet: PDCD10, SNAI2, E-cad, VIM and β- catenin used in this study were from following sources: antibodies against PDCD10 and GAPDH were purchased from Abcam and Utibody, respectively (ab110531 and UM4002, respectively); antibodies against SNAI2 and GAPDH were acquired from Affinity (AF4002) and Utibody (UM4001), respectively; antibodies against E-cad, VIM, and β- catenin (20874-1-AP, 22018-1-AP and 10366-1-AP, respectively) were obtained from Proteintech.

    Techniques: Migration, Expressing, Western Blot, Transduction, Plasmid Preparation, Labeling, Immunofluorescence, Two Tailed Test

    miR-222-3p targets PDCD10 to repress cell migration by downregulating the Wnt/ β -catenin signaling pathway. (A) qPCR analysis of β -catenin and TCF4 expression in HO 8910 PM cells (PDCD10-overexpressing groups) and SKOV3 cells (PDCD10-silenced groups). ( B ) Activity of the Wnt/ β -catenin signaling pathway in HEK-293T cells detected by the TOP flash/FOP flash dual-luciferase reporting system. HEK-293T cells were transfected with TOP flash/FOP flash plasmid and pRL-SV40 vector followed by transfection with or without PDCD10, and siRNA-NC/PDCD10, siRNA-03/PDCD10, or siRNA-04. Firefly luciferase activities were tested by a luminometer, and the activity of the Wnt/ β -catenin signaling pathway was recorded as TOP/FOP. ( C ) Western blot analysis of PDCD10 and β -catenin protein expression following overexpression of PDCD10 in HO 8910 PM cell fractions. β -actin was utilized as a marker for the cytosolic fractions. ( D ) HO 8910 PM cells transduced with or without PDCD10 were subjected to immunofluorescence with human-specific β -catenin antibodies. ( β -catenin in red). Bar, 10 µm. ( E ) β -catenin nuclei-intensity (detected by Operetta High-Content Imaging System (Perkin Elmer) in HO 8910 PM cell). ( F ) Western blot analysis of PDCD10, β -catenin translation levels in HO 8910 PM cells, or after coculturing with miR-ctrl mimic or miR-222-3p mimic in the presence or absence of PDCD10 (Left). The relative expression rate was calculated by Image J (Right). Data (A-B and E-F) represent the mean ± SD in different assays (n=3), *, P< 0.05; **, P< 0.01, determined by unpaired two-tailed t-test.

    Journal: Theranostics

    Article Title: Non-canonical signaling pathway of SNAI2 induces EMT in ovarian cancer cells by suppressing miR-222-3p transcription and upregulating PDCD10

    doi: 10.7150/thno.43198

    Figure Lengend Snippet: miR-222-3p targets PDCD10 to repress cell migration by downregulating the Wnt/ β -catenin signaling pathway. (A) qPCR analysis of β -catenin and TCF4 expression in HO 8910 PM cells (PDCD10-overexpressing groups) and SKOV3 cells (PDCD10-silenced groups). ( B ) Activity of the Wnt/ β -catenin signaling pathway in HEK-293T cells detected by the TOP flash/FOP flash dual-luciferase reporting system. HEK-293T cells were transfected with TOP flash/FOP flash plasmid and pRL-SV40 vector followed by transfection with or without PDCD10, and siRNA-NC/PDCD10, siRNA-03/PDCD10, or siRNA-04. Firefly luciferase activities were tested by a luminometer, and the activity of the Wnt/ β -catenin signaling pathway was recorded as TOP/FOP. ( C ) Western blot analysis of PDCD10 and β -catenin protein expression following overexpression of PDCD10 in HO 8910 PM cell fractions. β -actin was utilized as a marker for the cytosolic fractions. ( D ) HO 8910 PM cells transduced with or without PDCD10 were subjected to immunofluorescence with human-specific β -catenin antibodies. ( β -catenin in red). Bar, 10 µm. ( E ) β -catenin nuclei-intensity (detected by Operetta High-Content Imaging System (Perkin Elmer) in HO 8910 PM cell). ( F ) Western blot analysis of PDCD10, β -catenin translation levels in HO 8910 PM cells, or after coculturing with miR-ctrl mimic or miR-222-3p mimic in the presence or absence of PDCD10 (Left). The relative expression rate was calculated by Image J (Right). Data (A-B and E-F) represent the mean ± SD in different assays (n=3), *, P< 0.05; **, P< 0.01, determined by unpaired two-tailed t-test.

    Article Snippet: PDCD10, SNAI2, E-cad, VIM and β- catenin used in this study were from following sources: antibodies against PDCD10 and GAPDH were purchased from Abcam and Utibody, respectively (ab110531 and UM4002, respectively); antibodies against SNAI2 and GAPDH were acquired from Affinity (AF4002) and Utibody (UM4001), respectively; antibodies against E-cad, VIM, and β- catenin (20874-1-AP, 22018-1-AP and 10366-1-AP, respectively) were obtained from Proteintech.

    Techniques: Migration, Expressing, Activity Assay, Luciferase, Transfection, Plasmid Preparation, Western Blot, Over Expression, Marker, Transduction, Immunofluorescence, Imaging, Two Tailed Test

    SNAI2 increases cell migration via the SNAI2/miR-222-3p/PDCD10 axis and PDCD10-mediated promotion of EMT and Wnt/ β- catenin signaling. ( A ) Pearson's correlation scatter plots showed the fold changes of PDCD10 mRNA, miR-222-3p miRNA and SNAI2 mRNA levels in EOC tissues (n=38). ( B ) SNAI2 could enhance PDCD10 expression. PDCD10 mRNA expression levels were up-regulated after SNAI2 was overexpressed, but were down-regulated after SNAI2 was knocked down. ( C ) After transfection of SNAI2-overexpressing vectors and SNAI2 knockdown vectors (SNAI2 shRNA-01 and SNAI2 shRNA-02), the expression of PDCD10 was tested by Western blot in HO 8910 PM and SKOV3 cells. ( D ) Spearman correlation analysis of PDCD10, CDH1, VIM, SNAI2 and CTNNB1 levels in OC tissues (n=597) from the TCGA datasets. ( E ) A working model describing the interaction between SNAI2/miR-222-3p/PDCD10 during cancer metastasis. PDCD10 was critical for EOC cell mesenchymal movement and cell survival by maintaining low cell adhesion and high Wnt signaling. SNAI2 regulated PDCD10 by inhibiting pri-miR-222-3p expression and subsequent targeting of genes. SNAI2 regulated PDCD10 by inhibiting miR-222-3p expression, and further activated the downstream EMT signaling and Wnt/ β- catenin signaling to promote the expression of VIM, and β- catenin. These changes contributed to the EOC cell formation and migration, ultimately increasing tumor formation and metastasis. The data (B) represent the mean ± SD in different assays (n=3), **, P< 0.01; ***, P< 0.001, determined by unpaired two-tailed t-test.

    Journal: Theranostics

    Article Title: Non-canonical signaling pathway of SNAI2 induces EMT in ovarian cancer cells by suppressing miR-222-3p transcription and upregulating PDCD10

    doi: 10.7150/thno.43198

    Figure Lengend Snippet: SNAI2 increases cell migration via the SNAI2/miR-222-3p/PDCD10 axis and PDCD10-mediated promotion of EMT and Wnt/ β- catenin signaling. ( A ) Pearson's correlation scatter plots showed the fold changes of PDCD10 mRNA, miR-222-3p miRNA and SNAI2 mRNA levels in EOC tissues (n=38). ( B ) SNAI2 could enhance PDCD10 expression. PDCD10 mRNA expression levels were up-regulated after SNAI2 was overexpressed, but were down-regulated after SNAI2 was knocked down. ( C ) After transfection of SNAI2-overexpressing vectors and SNAI2 knockdown vectors (SNAI2 shRNA-01 and SNAI2 shRNA-02), the expression of PDCD10 was tested by Western blot in HO 8910 PM and SKOV3 cells. ( D ) Spearman correlation analysis of PDCD10, CDH1, VIM, SNAI2 and CTNNB1 levels in OC tissues (n=597) from the TCGA datasets. ( E ) A working model describing the interaction between SNAI2/miR-222-3p/PDCD10 during cancer metastasis. PDCD10 was critical for EOC cell mesenchymal movement and cell survival by maintaining low cell adhesion and high Wnt signaling. SNAI2 regulated PDCD10 by inhibiting pri-miR-222-3p expression and subsequent targeting of genes. SNAI2 regulated PDCD10 by inhibiting miR-222-3p expression, and further activated the downstream EMT signaling and Wnt/ β- catenin signaling to promote the expression of VIM, and β- catenin. These changes contributed to the EOC cell formation and migration, ultimately increasing tumor formation and metastasis. The data (B) represent the mean ± SD in different assays (n=3), **, P< 0.01; ***, P< 0.001, determined by unpaired two-tailed t-test.

    Article Snippet: PDCD10, SNAI2, E-cad, VIM and β- catenin used in this study were from following sources: antibodies against PDCD10 and GAPDH were purchased from Abcam and Utibody, respectively (ab110531 and UM4002, respectively); antibodies against SNAI2 and GAPDH were acquired from Affinity (AF4002) and Utibody (UM4001), respectively; antibodies against E-cad, VIM, and β- catenin (20874-1-AP, 22018-1-AP and 10366-1-AP, respectively) were obtained from Proteintech.

    Techniques: Migration, Expressing, Transfection, shRNA, Western Blot, Two Tailed Test

    Expression of PDCD10 in human pituitary adenomas. ( A ) A dataset (GSE26966) was obtained from the GEO database to compare the mRNA expression level of PDCD10 between PA tumor tissues (N=14) and normal pituitary tissue (N=9). ( B ) Relative mRNA expression levels of PDCD10 by RT-qPCR in invasive (n=15) and non-invasive (n=15) pituitary adenomas. Non-invasive values were set to 1. ( C ) Representative Western blots showed the expression level of PCDC10 protein (25 kDa) in invasive and noninvasive pituitary adenomas. Band intensities were quantified and normalized to GAPDH. ( D ) Representative images of immunohistochemistry evaluated the expression of PDCD10 in invasive and non-invasive (n=15) pituitary adenomas. **P < 0.01; ***P < 0.001; ****P < 0.0001.

    Journal: Aging (Albany NY)

    Article Title: PDCD10 promotes the aggressive behaviors of pituitary adenomas by up-regulating CXCR2 and activating downstream AKT/ERK signaling

    doi: 10.18632/aging.204206

    Figure Lengend Snippet: Expression of PDCD10 in human pituitary adenomas. ( A ) A dataset (GSE26966) was obtained from the GEO database to compare the mRNA expression level of PDCD10 between PA tumor tissues (N=14) and normal pituitary tissue (N=9). ( B ) Relative mRNA expression levels of PDCD10 by RT-qPCR in invasive (n=15) and non-invasive (n=15) pituitary adenomas. Non-invasive values were set to 1. ( C ) Representative Western blots showed the expression level of PCDC10 protein (25 kDa) in invasive and noninvasive pituitary adenomas. Band intensities were quantified and normalized to GAPDH. ( D ) Representative images of immunohistochemistry evaluated the expression of PDCD10 in invasive and non-invasive (n=15) pituitary adenomas. **P < 0.01; ***P < 0.001; ****P < 0.0001.

    Article Snippet: Antibodies to PDCD10, E-cadherin, N-cadherin and Vimentin (VIM) were purchased from Proteintech Group.

    Techniques: Expressing, Quantitative RT-PCR, Western Blot, Immunohistochemistry

    PDCD10 silencing suppresses the proliferation, migration, invasion and EMT of PA cells. ( A ) Western blotting was performed to detect the impact of PDCD10 silencing on the expression of EMT markers in Att-20 cells and TtT/GF cells. ( B ) Band intensities were quantified and normalized to GAPDH. ( C , D ) CCK-8 assay was used to assess cell proliferation capacity after PDCD10 silencing in Att-20 and TtT/GF cells. ( E , F ) Scratch assay was used to examine the relative migration rates of Att-20 and TtT/GF cells (magnification:100x). ( G , H ) Transwell invasion assay was used to analyze the invasion potential of Att-20 and TtT/GF cells (magnification: 200x). * P < 0.05.

    Journal: Aging (Albany NY)

    Article Title: PDCD10 promotes the aggressive behaviors of pituitary adenomas by up-regulating CXCR2 and activating downstream AKT/ERK signaling

    doi: 10.18632/aging.204206

    Figure Lengend Snippet: PDCD10 silencing suppresses the proliferation, migration, invasion and EMT of PA cells. ( A ) Western blotting was performed to detect the impact of PDCD10 silencing on the expression of EMT markers in Att-20 cells and TtT/GF cells. ( B ) Band intensities were quantified and normalized to GAPDH. ( C , D ) CCK-8 assay was used to assess cell proliferation capacity after PDCD10 silencing in Att-20 and TtT/GF cells. ( E , F ) Scratch assay was used to examine the relative migration rates of Att-20 and TtT/GF cells (magnification:100x). ( G , H ) Transwell invasion assay was used to analyze the invasion potential of Att-20 and TtT/GF cells (magnification: 200x). * P < 0.05.

    Article Snippet: Antibodies to PDCD10, E-cadherin, N-cadherin and Vimentin (VIM) were purchased from Proteintech Group.

    Techniques: Migration, Western Blot, Expressing, CCK-8 Assay, Wound Healing Assay, Transwell Invasion Assay

    Overexpression of PDCD10 promotes the proliferation, migration, invasion and EMT of PA cells. ( A , B ) Western blotting was performed to detect the impact of PDCD10 overexpression on the expression levels of EMT markers in Att-20 cells and TtT/GF cells. Band intensities were quantified and normalized to GAPDH. ( C , D ) CCK-8 assay was used to assess cell proliferation potential after PDCD10 overexpression in Att-20 and TtT/GF cells. ( E , F ) Scratch assay was employed to examine the relative migration rates of Att-20 and TtT/GF cells after PDCD10 overexpression (magnification:100x). ( G , H ) Transwell invasion assay was used to detect the invasion potential of Att-20 and TtT/GF cells after PDCD10 overexpression (magnification: 200x). * P < 0.05.

    Journal: Aging (Albany NY)

    Article Title: PDCD10 promotes the aggressive behaviors of pituitary adenomas by up-regulating CXCR2 and activating downstream AKT/ERK signaling

    doi: 10.18632/aging.204206

    Figure Lengend Snippet: Overexpression of PDCD10 promotes the proliferation, migration, invasion and EMT of PA cells. ( A , B ) Western blotting was performed to detect the impact of PDCD10 overexpression on the expression levels of EMT markers in Att-20 cells and TtT/GF cells. Band intensities were quantified and normalized to GAPDH. ( C , D ) CCK-8 assay was used to assess cell proliferation potential after PDCD10 overexpression in Att-20 and TtT/GF cells. ( E , F ) Scratch assay was employed to examine the relative migration rates of Att-20 and TtT/GF cells after PDCD10 overexpression (magnification:100x). ( G , H ) Transwell invasion assay was used to detect the invasion potential of Att-20 and TtT/GF cells after PDCD10 overexpression (magnification: 200x). * P < 0.05.

    Article Snippet: Antibodies to PDCD10, E-cadherin, N-cadherin and Vimentin (VIM) were purchased from Proteintech Group.

    Techniques: Over Expression, Migration, Western Blot, Expressing, CCK-8 Assay, Wound Healing Assay, Transwell Invasion Assay

    PDCD10 alters the protein expression level of CXCR2 and regulates the activation of downstream AKT/ERK signal pathways. ( A , B ) Western blotting was used to detect the expression level of CXCR2 and phosphorylation level of AKT, ERK1/2 and STAT3 in Att-20 cells after PDCD10 silencing or overexpression. Band intensities were quantified and normalized to GAPDH. * P < 0.05. ( C , D ) In TtT/GF cells, western blotting was performed to examine the expression level of CXCR2 and phosphorylation level of AKT, ERK1/2 and STAT3 after PDCD10 silencing or overexpression. Band intensities were quantified and normalized to GAPDH. * P < 0.05.

    Journal: Aging (Albany NY)

    Article Title: PDCD10 promotes the aggressive behaviors of pituitary adenomas by up-regulating CXCR2 and activating downstream AKT/ERK signaling

    doi: 10.18632/aging.204206

    Figure Lengend Snippet: PDCD10 alters the protein expression level of CXCR2 and regulates the activation of downstream AKT/ERK signal pathways. ( A , B ) Western blotting was used to detect the expression level of CXCR2 and phosphorylation level of AKT, ERK1/2 and STAT3 in Att-20 cells after PDCD10 silencing or overexpression. Band intensities were quantified and normalized to GAPDH. * P < 0.05. ( C , D ) In TtT/GF cells, western blotting was performed to examine the expression level of CXCR2 and phosphorylation level of AKT, ERK1/2 and STAT3 after PDCD10 silencing or overexpression. Band intensities were quantified and normalized to GAPDH. * P < 0.05.

    Article Snippet: Antibodies to PDCD10, E-cadherin, N-cadherin and Vimentin (VIM) were purchased from Proteintech Group.

    Techniques: Expressing, Activation Assay, Western Blot, Over Expression

    Activation of CXCR2 rescues the inactivation of AKT/ERK signaling and the tumor-suppressive effects induced by PDCD10 silencing. ( A , B ) Western blotting was performed to examine the phosphorylation levels of AKT and ERK1/2 after recombinant CXCL2 administration (200ng/ml) in Att-20 and TtT/GF cells with PDCD10 silencing. ( C , D ) CCK-8 assay was used to evaluate cell proliferation potential after CXCL2 administration in Att-20 and TtT/GF cells with PDCD10 silencing. ( E , F ) Relative migration rate of ATT-20 and TtT/GF cells with PDCD10 silencing was analyzed by scratch assay after CXCL2 administration (magnification: 100x). ( G , H ) Transwell invasion assay was employed to assess the invasion capacity of Att-20 and TtT/GF cells with PDCD10 silencing after CXCL2 administration (magnification: 200x). * P < 0.05.

    Journal: Aging (Albany NY)

    Article Title: PDCD10 promotes the aggressive behaviors of pituitary adenomas by up-regulating CXCR2 and activating downstream AKT/ERK signaling

    doi: 10.18632/aging.204206

    Figure Lengend Snippet: Activation of CXCR2 rescues the inactivation of AKT/ERK signaling and the tumor-suppressive effects induced by PDCD10 silencing. ( A , B ) Western blotting was performed to examine the phosphorylation levels of AKT and ERK1/2 after recombinant CXCL2 administration (200ng/ml) in Att-20 and TtT/GF cells with PDCD10 silencing. ( C , D ) CCK-8 assay was used to evaluate cell proliferation potential after CXCL2 administration in Att-20 and TtT/GF cells with PDCD10 silencing. ( E , F ) Relative migration rate of ATT-20 and TtT/GF cells with PDCD10 silencing was analyzed by scratch assay after CXCL2 administration (magnification: 100x). ( G , H ) Transwell invasion assay was employed to assess the invasion capacity of Att-20 and TtT/GF cells with PDCD10 silencing after CXCL2 administration (magnification: 200x). * P < 0.05.

    Article Snippet: Antibodies to PDCD10, E-cadherin, N-cadherin and Vimentin (VIM) were purchased from Proteintech Group.

    Techniques: Activation Assay, Western Blot, Recombinant, CCK-8 Assay, Migration, Wound Healing Assay, Transwell Invasion Assay

    PDCD10 silencing impairs the tumorigenesis and reduces CXCR2 expression of PA cells in vivo . ( A , B ) Images for Att-20 and TtT/GF xenografts from nude mice (Left). Statistical analysis of xenograft tumor weights (Right). ( C , D ) Representative IHC images of Att-20 and TtT/GF xenograft tumor tissues for PDCD10, CXCR2 and Ki-67 staining (200x). Scale bars: 100μm. ( E , F ) Intensity of PDCD10, CXCR2 and Ki-67 staining were analyzed by IHC-Profiler. ( G , H ) Western blotting was performed to examine the expression of CXCR2 in Att-20 and TtT/GF xenograft tumor samples. Band intensities were quantified and normalized to GAPDH. * P < 0.05.

    Journal: Aging (Albany NY)

    Article Title: PDCD10 promotes the aggressive behaviors of pituitary adenomas by up-regulating CXCR2 and activating downstream AKT/ERK signaling

    doi: 10.18632/aging.204206

    Figure Lengend Snippet: PDCD10 silencing impairs the tumorigenesis and reduces CXCR2 expression of PA cells in vivo . ( A , B ) Images for Att-20 and TtT/GF xenografts from nude mice (Left). Statistical analysis of xenograft tumor weights (Right). ( C , D ) Representative IHC images of Att-20 and TtT/GF xenograft tumor tissues for PDCD10, CXCR2 and Ki-67 staining (200x). Scale bars: 100μm. ( E , F ) Intensity of PDCD10, CXCR2 and Ki-67 staining were analyzed by IHC-Profiler. ( G , H ) Western blotting was performed to examine the expression of CXCR2 in Att-20 and TtT/GF xenograft tumor samples. Band intensities were quantified and normalized to GAPDH. * P < 0.05.

    Article Snippet: Antibodies to PDCD10, E-cadherin, N-cadherin and Vimentin (VIM) were purchased from Proteintech Group.

    Techniques: Expressing, In Vivo, Staining, Western Blot

    Murepavadin induces intracellular Ca 2+ mobilization, degranulation and chemokine production in MCs through MRGPRX2. (A) Calcium mobilization measurement following murepavadin (10 μM) stimulation of Fura-2 loaded WT-RBL and RBL-MRGPRX2 cells. (B) WT-RBL and RBL-MRGPRX2 cells were exposed to 10 μM murepavadin (30 min), and degranulation was assayed by measuring the release of β-hexosaminidase. (C) RBL-MRGPRX2 cells were incubated in the presence or absence of pertussis toxin (PTx), at a concentration of 100 ng/mL for 16 h and exposed to indicated concentrations of murepavadin (30 min) and degranulation was assayed by measuring the release of β-hexosaminidase. (D, E) WT-RBL and RBL-MRGPRX2 cells were incubated in the presence or absence of pertussis toxin (PTx) at a concentration of 100 ng/mL for 16 h and exposed to 10 μM of murepavadin (24 h) and the production of cytokine TNF-α and chemokine CCL2 were quantified by ELISA. Data presented are the mean ± SEM of at least three experiments. Statistical significance was determined by two-way ANOVA with Šídák’s or Tukey’s multiple comparisons at a value * p < 0.05, *** p < 0.001, **** p < 0.0001, and ns denotes “not significant”.

    Journal: Frontiers in Immunology

    Article Title: Murepavadin, a Small Molecule Host Defense Peptide Mimetic, Activates Mast Cells via MRGPRX2 and MrgprB2

    doi: 10.3389/fimmu.2021.689410

    Figure Lengend Snippet: Murepavadin induces intracellular Ca 2+ mobilization, degranulation and chemokine production in MCs through MRGPRX2. (A) Calcium mobilization measurement following murepavadin (10 μM) stimulation of Fura-2 loaded WT-RBL and RBL-MRGPRX2 cells. (B) WT-RBL and RBL-MRGPRX2 cells were exposed to 10 μM murepavadin (30 min), and degranulation was assayed by measuring the release of β-hexosaminidase. (C) RBL-MRGPRX2 cells were incubated in the presence or absence of pertussis toxin (PTx), at a concentration of 100 ng/mL for 16 h and exposed to indicated concentrations of murepavadin (30 min) and degranulation was assayed by measuring the release of β-hexosaminidase. (D, E) WT-RBL and RBL-MRGPRX2 cells were incubated in the presence or absence of pertussis toxin (PTx) at a concentration of 100 ng/mL for 16 h and exposed to 10 μM of murepavadin (24 h) and the production of cytokine TNF-α and chemokine CCL2 were quantified by ELISA. Data presented are the mean ± SEM of at least three experiments. Statistical significance was determined by two-way ANOVA with Šídák’s or Tukey’s multiple comparisons at a value * p < 0.05, *** p < 0.001, **** p < 0.0001, and ns denotes “not significant”.

    Article Snippet: MRGPRX2-Tango plasmid (Addgene no. 66440) was a gift from Dr. Bryan Roth.

    Techniques: Incubation, Concentration Assay, Enzyme-linked Immunosorbent Assay

    Naturally occurring MRGPRX2 missense variants are hypo-responsive to murepavadin for MC degranulation. (A) Snake diagram of MRGPRX2 indicating the two amino acid residues to be investigated. (B) Single amino acid substitution for each of the MRGPRX2 variant is shown in the table. (C) Cell surface receptor expression of the wild-type MRGPRX2 (WT) and its missense variants (G165E and V282M) was confirmed using flow cytometry. (D) RBL cells expressing MRGPRX2 (WT) and its variants (G165E and V282M) were exposed to 10 μM murepavadin (30 min) and degranulation was assayed by measuring the release of β-hexosaminidase. Statistical significance was determined by two-way ANOVA with Tukey’s multiple comparisons at a value ** p < 0.01 and **** p < 0.0001.

    Journal: Frontiers in Immunology

    Article Title: Murepavadin, a Small Molecule Host Defense Peptide Mimetic, Activates Mast Cells via MRGPRX2 and MrgprB2

    doi: 10.3389/fimmu.2021.689410

    Figure Lengend Snippet: Naturally occurring MRGPRX2 missense variants are hypo-responsive to murepavadin for MC degranulation. (A) Snake diagram of MRGPRX2 indicating the two amino acid residues to be investigated. (B) Single amino acid substitution for each of the MRGPRX2 variant is shown in the table. (C) Cell surface receptor expression of the wild-type MRGPRX2 (WT) and its missense variants (G165E and V282M) was confirmed using flow cytometry. (D) RBL cells expressing MRGPRX2 (WT) and its variants (G165E and V282M) were exposed to 10 μM murepavadin (30 min) and degranulation was assayed by measuring the release of β-hexosaminidase. Statistical significance was determined by two-way ANOVA with Tukey’s multiple comparisons at a value ** p < 0.01 and **** p < 0.0001.

    Article Snippet: MRGPRX2-Tango plasmid (Addgene no. 66440) was a gift from Dr. Bryan Roth.

    Techniques: Variant Assay, Cell Surface Receptor Assay, Expressing, Flow Cytometry

    Murepavadin does not promote β-arrestin recruitment following MRGPRX2 activation. (A, B) HTLA-MRGPRX2 cells were exposed to indicated concentrations of C48/80 or murepavadin for 16 h. Medium was removed and Bright-Glo solution (100 µL) was added into each well (96-well plate) and β-arrestin gene expression (in relative luminescence unit) was measured. (C, D) HTLA-MRGPRX2 cells were exposed to MRGPRX2 ligand (C48/80 or murepavadin, 16 h) and cell surface receptor expression of MRGPRX2 was confirmed using flow cytometry and quantified using a mean fluorescent intensity (MFI) in comparison to the untreated control. (E, F) Representative histograms of MRGPRX2 cell surface receptor expression of HTLA-MRGPRX2 cells. Statistical significance was determined by one-way ANOVA with Dunnett’s multiple comparisons at a value **** p < 0.0001 and ns denotes “not significant”.

    Journal: Frontiers in Immunology

    Article Title: Murepavadin, a Small Molecule Host Defense Peptide Mimetic, Activates Mast Cells via MRGPRX2 and MrgprB2

    doi: 10.3389/fimmu.2021.689410

    Figure Lengend Snippet: Murepavadin does not promote β-arrestin recruitment following MRGPRX2 activation. (A, B) HTLA-MRGPRX2 cells were exposed to indicated concentrations of C48/80 or murepavadin for 16 h. Medium was removed and Bright-Glo solution (100 µL) was added into each well (96-well plate) and β-arrestin gene expression (in relative luminescence unit) was measured. (C, D) HTLA-MRGPRX2 cells were exposed to MRGPRX2 ligand (C48/80 or murepavadin, 16 h) and cell surface receptor expression of MRGPRX2 was confirmed using flow cytometry and quantified using a mean fluorescent intensity (MFI) in comparison to the untreated control. (E, F) Representative histograms of MRGPRX2 cell surface receptor expression of HTLA-MRGPRX2 cells. Statistical significance was determined by one-way ANOVA with Dunnett’s multiple comparisons at a value **** p < 0.0001 and ns denotes “not significant”.

    Article Snippet: MRGPRX2-Tango plasmid (Addgene no. 66440) was a gift from Dr. Bryan Roth.

    Techniques: Activation Assay, Expressing, Cell Surface Receptor Assay, Flow Cytometry

    Murepavadin does not induce MRGPRX2 internalization or desensitization. (A) RBL-MRGPRX2 cells were exposed to MRGPRX2 ligand (C48/80 3 μg/mL or murepavadin 10 μM, 16 h) and cell surface receptor expression of MRGPRX2 was confirmed using flow cytometry. A representative histogram of MRGPRX2 cell surface receptor expression is shown. (B) Quantitative analysis of cell surface receptor expression was calculated using a mean fluorescent intensity (MFI) in comparison to the untreated control. (C, D) Calcium mobilization measurements of RBL-MRGPRX2 cells exposed to MRGPRX2 ligand (C48/80 3 μg/mL or murepavadin 10 μM, 16 h) following C48/80 (3 μg/mL) or murepavadin (10 μM) stimulation, respectively. Statistical significance was determined by one-way ANOVA with Dunnett’s multiple comparisons and two-way ANOVA with Šídák’s multiple comparisons at a value * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, and ns denotes “not significant”.

    Journal: Frontiers in Immunology

    Article Title: Murepavadin, a Small Molecule Host Defense Peptide Mimetic, Activates Mast Cells via MRGPRX2 and MrgprB2

    doi: 10.3389/fimmu.2021.689410

    Figure Lengend Snippet: Murepavadin does not induce MRGPRX2 internalization or desensitization. (A) RBL-MRGPRX2 cells were exposed to MRGPRX2 ligand (C48/80 3 μg/mL or murepavadin 10 μM, 16 h) and cell surface receptor expression of MRGPRX2 was confirmed using flow cytometry. A representative histogram of MRGPRX2 cell surface receptor expression is shown. (B) Quantitative analysis of cell surface receptor expression was calculated using a mean fluorescent intensity (MFI) in comparison to the untreated control. (C, D) Calcium mobilization measurements of RBL-MRGPRX2 cells exposed to MRGPRX2 ligand (C48/80 3 μg/mL or murepavadin 10 μM, 16 h) following C48/80 (3 μg/mL) or murepavadin (10 μM) stimulation, respectively. Statistical significance was determined by one-way ANOVA with Dunnett’s multiple comparisons and two-way ANOVA with Šídák’s multiple comparisons at a value * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, and ns denotes “not significant”.

    Article Snippet: MRGPRX2-Tango plasmid (Addgene no. 66440) was a gift from Dr. Bryan Roth.

    Techniques: Cell Surface Receptor Assay, Expressing, Flow Cytometry

    Proposed mechanism of action of murepavadin/MRGPRX2-induced bacterial clearance and wound healing. Murepavadin activates MCs via MRGPRX2. An increase in intracellular calcium following murepavadin stimulation triggers MC degranulation and chemokine production (IL-8, CCL3, CCL2, and TNF-α). These MC mediators induce immune cell recruitment and subsequently contribute to host defense and wound healing.

    Journal: Frontiers in Immunology

    Article Title: Murepavadin, a Small Molecule Host Defense Peptide Mimetic, Activates Mast Cells via MRGPRX2 and MrgprB2

    doi: 10.3389/fimmu.2021.689410

    Figure Lengend Snippet: Proposed mechanism of action of murepavadin/MRGPRX2-induced bacterial clearance and wound healing. Murepavadin activates MCs via MRGPRX2. An increase in intracellular calcium following murepavadin stimulation triggers MC degranulation and chemokine production (IL-8, CCL3, CCL2, and TNF-α). These MC mediators induce immune cell recruitment and subsequently contribute to host defense and wound healing.

    Article Snippet: MRGPRX2-Tango plasmid (Addgene no. 66440) was a gift from Dr. Bryan Roth.

    Techniques:

    (A) LAD2 cells were stimulated with different concentrations of AG-30/5C (0.01 – 1.5 μM) for 30 min. Percent degranulation (β-hexosaminidase release) was determined. (B) LAD2 cells were stably transduced with scrambled control shRNA or lentivirus shRNA targeting human MRGPRX2. Western blotting was performed to determine MRGPRX2 expression. β-actin was used as a loading control. (C) Control and MRGPRX2 knockdown LAD2 cells were stimulated with AG-30/5C (1 μM) and degranulation was determine. (D) RBL-2H3 and RBL-2H3 cell stably expressing MRGPRX2 (RBL-MRGPRX2) were stimulated with different concentrations of AG-30/5C (0.01 – 1.5 μM) and percent degranulation was determined. All data are expressed as a mean ± SEM of three experiments. Statistical significance was determined by non-parametric t-Test and two way ANOVA (P value<.05). Western blotting data presented are representative of three similar experiments. *** indicates P value<.001,** indicates P value <.01 and * indicates P value <.05.

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Angiogenic Host Defense Peptide AG-30/5C and Bradykinin B 2 receptor antagonist Icatibant are G protein-biased agonists for MRGPRX2 in mast cells

    doi: 10.4049/jimmunol.1801227

    Figure Lengend Snippet: (A) LAD2 cells were stimulated with different concentrations of AG-30/5C (0.01 – 1.5 μM) for 30 min. Percent degranulation (β-hexosaminidase release) was determined. (B) LAD2 cells were stably transduced with scrambled control shRNA or lentivirus shRNA targeting human MRGPRX2. Western blotting was performed to determine MRGPRX2 expression. β-actin was used as a loading control. (C) Control and MRGPRX2 knockdown LAD2 cells were stimulated with AG-30/5C (1 μM) and degranulation was determine. (D) RBL-2H3 and RBL-2H3 cell stably expressing MRGPRX2 (RBL-MRGPRX2) were stimulated with different concentrations of AG-30/5C (0.01 – 1.5 μM) and percent degranulation was determined. All data are expressed as a mean ± SEM of three experiments. Statistical significance was determined by non-parametric t-Test and two way ANOVA (P value<.05). Western blotting data presented are representative of three similar experiments. *** indicates P value<.001,** indicates P value <.01 and * indicates P value <.05.

    Article Snippet: MRGPRX2-Tango (Addgene #66440) and MRGPRX4-Tango (Addgene#66442) plasmids were gifts from Bryan Roth ( 32 ).

    Techniques: Stable Transfection, Transduction, shRNA, Western Blot, Expressing

    (A) RBL-MRGPRX2 cells cultured in absence (-PTx) or presence of pertussis toxin (+PTx, 100 ng/ml, 16h). Cells were stimulated with compound 48/80 (1μg/mL) or AG-30/5C (1 μM) and percent degranulation was determined. (B-C) RBL-MRGPRX2 cells were cultured in the absence or presence of PTx, loaded with Indo-1 (1 μM, 30 min) and stimulated with either compound 48/80 (1 μg/mL) or AG-30/5C (1 μM) as indicated by arrows and time course of calcium mobilization was determined. Representative traces of 3 similar experiments are shown. All data points for degranulation are expressed as mean ± SEM of three experiments. Statistical significance was determined by non-parametric t-Test and two way ANOVA (P value<.05). *** indicates P value<.001,** indicates P value <.01 and * indicates P value <.05.

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Angiogenic Host Defense Peptide AG-30/5C and Bradykinin B 2 receptor antagonist Icatibant are G protein-biased agonists for MRGPRX2 in mast cells

    doi: 10.4049/jimmunol.1801227

    Figure Lengend Snippet: (A) RBL-MRGPRX2 cells cultured in absence (-PTx) or presence of pertussis toxin (+PTx, 100 ng/ml, 16h). Cells were stimulated with compound 48/80 (1μg/mL) or AG-30/5C (1 μM) and percent degranulation was determined. (B-C) RBL-MRGPRX2 cells were cultured in the absence or presence of PTx, loaded with Indo-1 (1 μM, 30 min) and stimulated with either compound 48/80 (1 μg/mL) or AG-30/5C (1 μM) as indicated by arrows and time course of calcium mobilization was determined. Representative traces of 3 similar experiments are shown. All data points for degranulation are expressed as mean ± SEM of three experiments. Statistical significance was determined by non-parametric t-Test and two way ANOVA (P value<.05). *** indicates P value<.001,** indicates P value <.01 and * indicates P value <.05.

    Article Snippet: MRGPRX2-Tango (Addgene #66440) and MRGPRX4-Tango (Addgene#66442) plasmids were gifts from Bryan Roth ( 32 ).

    Techniques: Cell Culture

    (A) Modular design of Tango constructs. (B) Upon activation (1), β-arrestin is recruited to the C terminus of the receptor (2). This is followed by cleavage of the GPCR fusion protein at the TEV protease–cleavage site (3). Cleavage results in the release of the tTA transcription factor (4), which after transport to the nucleus activates transcription of the luciferase reporter gene (5). (C) Untransfected (HTLA) and stably transfected HTLA-MRGPRX2-Tango (HTLA-MRGPRX2) cells were incubated with anti-FLAG-PE antibody and cell surface receptor expression was determined by flow cytometry. A representative overlay histogram of MRGPRX2 expression is shown. (D) HTLA-MRGPRX2 cells were exposed to compound 48/80 (1.0 or 30 μg/mL) and AG-30/5C (1.0 and 10 μM) respectively and β-arrestin-mediated gene expression was determined. (E) Representative Ca2+ measurement traces for compound 48/80 (1 μg/mL and 30 μg/mL) and AG-30/5C (1 μM and 10 μM) are shown. All data points for the β-arrestin activation assay are expressed as a mean ± SEM of three experiments. Statistical significance was determined by non-parametric t-Test and two way ANOVA (P value<.05). *** indicates P value<.001,** indicates P value <.01 and * indicates P value <.05.

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Angiogenic Host Defense Peptide AG-30/5C and Bradykinin B 2 receptor antagonist Icatibant are G protein-biased agonists for MRGPRX2 in mast cells

    doi: 10.4049/jimmunol.1801227

    Figure Lengend Snippet: (A) Modular design of Tango constructs. (B) Upon activation (1), β-arrestin is recruited to the C terminus of the receptor (2). This is followed by cleavage of the GPCR fusion protein at the TEV protease–cleavage site (3). Cleavage results in the release of the tTA transcription factor (4), which after transport to the nucleus activates transcription of the luciferase reporter gene (5). (C) Untransfected (HTLA) and stably transfected HTLA-MRGPRX2-Tango (HTLA-MRGPRX2) cells were incubated with anti-FLAG-PE antibody and cell surface receptor expression was determined by flow cytometry. A representative overlay histogram of MRGPRX2 expression is shown. (D) HTLA-MRGPRX2 cells were exposed to compound 48/80 (1.0 or 30 μg/mL) and AG-30/5C (1.0 and 10 μM) respectively and β-arrestin-mediated gene expression was determined. (E) Representative Ca2+ measurement traces for compound 48/80 (1 μg/mL and 30 μg/mL) and AG-30/5C (1 μM and 10 μM) are shown. All data points for the β-arrestin activation assay are expressed as a mean ± SEM of three experiments. Statistical significance was determined by non-parametric t-Test and two way ANOVA (P value<.05). *** indicates P value<.001,** indicates P value <.01 and * indicates P value <.05.

    Article Snippet: MRGPRX2-Tango (Addgene #66440) and MRGPRX4-Tango (Addgene#66442) plasmids were gifts from Bryan Roth ( 32 ).

    Techniques: Construct, Activation Assay, Luciferase, Stable Transfection, Transfection, Incubation, Cell Surface Receptor Assay, Expressing, Flow Cytometry

    (A) RBL-MRGPRX2 cells (0.5 × 106) were cultured in the absence (unstimulated) or presence of compound 48/80 (1 μg/mL) or AG-30/5C (1 μM) for 16 h and cells surface expression of MRGPRX2 was determined by flow cytometry using anti-MRGPRX2-PE antibody. The data is presented as mean fluorescent intensity (MFI) of three experiments (B) A representative histogram of cell surface MRGPRX2 receptor expression is shown. (C) LAD2 cells were cultured in the absence (Control) or presence of in compound 48/80 (1 μg/mL) or AG-30/5C (1 μM) for 16h, washed and plated in a 96 well plate (10,000 cells/well). Cells were stimulated with compound 48/80 (1.0 μg/mL) and AG-30/5C (1 μM) respectively for 30 min and percent degranulation was determined by β-hexosaminidase release assay. All the points are expressed as a mean ± SEM of three experiments. Statistical significance was determined by non-parametric t-Test and two way ANOVA. *** indicates P value<.001,** indicates P value <.01 and * indicates P value <.05.

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Angiogenic Host Defense Peptide AG-30/5C and Bradykinin B 2 receptor antagonist Icatibant are G protein-biased agonists for MRGPRX2 in mast cells

    doi: 10.4049/jimmunol.1801227

    Figure Lengend Snippet: (A) RBL-MRGPRX2 cells (0.5 × 106) were cultured in the absence (unstimulated) or presence of compound 48/80 (1 μg/mL) or AG-30/5C (1 μM) for 16 h and cells surface expression of MRGPRX2 was determined by flow cytometry using anti-MRGPRX2-PE antibody. The data is presented as mean fluorescent intensity (MFI) of three experiments (B) A representative histogram of cell surface MRGPRX2 receptor expression is shown. (C) LAD2 cells were cultured in the absence (Control) or presence of in compound 48/80 (1 μg/mL) or AG-30/5C (1 μM) for 16h, washed and plated in a 96 well plate (10,000 cells/well). Cells were stimulated with compound 48/80 (1.0 μg/mL) and AG-30/5C (1 μM) respectively for 30 min and percent degranulation was determined by β-hexosaminidase release assay. All the points are expressed as a mean ± SEM of three experiments. Statistical significance was determined by non-parametric t-Test and two way ANOVA. *** indicates P value<.001,** indicates P value <.01 and * indicates P value <.05.

    Article Snippet: MRGPRX2-Tango (Addgene #66440) and MRGPRX4-Tango (Addgene#66442) plasmids were gifts from Bryan Roth ( 32 ).

    Techniques: Cell Culture, Expressing, Flow Cytometry, Release Assay

    (A) HTLA cells stably expressing MRGPRX2-Tango were incubated with resveratrol (100 μM, 5 min) followed by overnight stimulation with compound 48/80 (3.0, 10 and 20 μg/mL) or AG-30/5C (10 μM) respectively and relative luminescence was determined. (B) RBL-MRGPRX2 cells and (C) LAD2 cells either left untreated (Control) or incubated with resveratrol (100 μM, 5 min) and stimulated with compound 48/80 (1μg/mL) or AG-30/5C (1 μM) for 30 min and percent degranulation was determined. Statistical significance was determined by non-parametric t-Test and two way ANOVA. *** indicates P value<.001,** indicates P value <.01 and * indicates P value <.05.

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Angiogenic Host Defense Peptide AG-30/5C and Bradykinin B 2 receptor antagonist Icatibant are G protein-biased agonists for MRGPRX2 in mast cells

    doi: 10.4049/jimmunol.1801227

    Figure Lengend Snippet: (A) HTLA cells stably expressing MRGPRX2-Tango were incubated with resveratrol (100 μM, 5 min) followed by overnight stimulation with compound 48/80 (3.0, 10 and 20 μg/mL) or AG-30/5C (10 μM) respectively and relative luminescence was determined. (B) RBL-MRGPRX2 cells and (C) LAD2 cells either left untreated (Control) or incubated with resveratrol (100 μM, 5 min) and stimulated with compound 48/80 (1μg/mL) or AG-30/5C (1 μM) for 30 min and percent degranulation was determined. Statistical significance was determined by non-parametric t-Test and two way ANOVA. *** indicates P value<.001,** indicates P value <.01 and * indicates P value <.05.

    Article Snippet: MRGPRX2-Tango (Addgene #66440) and MRGPRX4-Tango (Addgene#66442) plasmids were gifts from Bryan Roth ( 32 ).

    Techniques: Stable Transfection, Expressing, Incubation

    (A and B) RBL-MRGPRX2 cells were cultured in the absence or presence of PTx (100 ng/mL, 16 h) and percent degranulation and Ca2+ mobilization in response to Icatibant (20 μg/mL) was determined as described for compound 48/80 and AG-30/5C (Figure 2). (C) RBL-MRGPRX2 and (D) LAD2 cells were incubated with resveratrol (100 μM, 5 min), stimulated with Icatibant (20 μg/mL) for 30 min and percent degranulation was determined. (E) HTLA cells expressing MRGPRX2 (HTLA-MRGPRX2) were exposed to medium (Control) or resveratrol (100 μM of 5 min) followed by overnight stimulation with compound 48/80 (10 μg/mL) or Icatibant (30 μg/mL) and chemiluminescence was measured (F) Representative Ca2+ traces for compound 48/80 (30 μg/mL) and Icatibant (30 μg/mL) in HTLA-MRGPRX2 cells are shown. Statistical significance was determined by non-parametric t-Test and two way ANOVA. *** indicates P value<.001,** indicates P value <.01 and * indicates P value <.05.

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Angiogenic Host Defense Peptide AG-30/5C and Bradykinin B 2 receptor antagonist Icatibant are G protein-biased agonists for MRGPRX2 in mast cells

    doi: 10.4049/jimmunol.1801227

    Figure Lengend Snippet: (A and B) RBL-MRGPRX2 cells were cultured in the absence or presence of PTx (100 ng/mL, 16 h) and percent degranulation and Ca2+ mobilization in response to Icatibant (20 μg/mL) was determined as described for compound 48/80 and AG-30/5C (Figure 2). (C) RBL-MRGPRX2 and (D) LAD2 cells were incubated with resveratrol (100 μM, 5 min), stimulated with Icatibant (20 μg/mL) for 30 min and percent degranulation was determined. (E) HTLA cells expressing MRGPRX2 (HTLA-MRGPRX2) were exposed to medium (Control) or resveratrol (100 μM of 5 min) followed by overnight stimulation with compound 48/80 (10 μg/mL) or Icatibant (30 μg/mL) and chemiluminescence was measured (F) Representative Ca2+ traces for compound 48/80 (30 μg/mL) and Icatibant (30 μg/mL) in HTLA-MRGPRX2 cells are shown. Statistical significance was determined by non-parametric t-Test and two way ANOVA. *** indicates P value<.001,** indicates P value <.01 and * indicates P value <.05.

    Article Snippet: MRGPRX2-Tango (Addgene #66440) and MRGPRX4-Tango (Addgene#66442) plasmids were gifts from Bryan Roth ( 32 ).

    Techniques: Cell Culture, Incubation, Expressing

    We propose that in addition to its direct antimicrobial activity, AG-30/5C contributed to host defense via MC degranulation through MRGPRX2. AG-30/5C contributes to wound healing via its action on vascular endothelial cells keratinocyte proliferation and migration through MRGPRX3 and MRGPRX4.

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Angiogenic Host Defense Peptide AG-30/5C and Bradykinin B 2 receptor antagonist Icatibant are G protein-biased agonists for MRGPRX2 in mast cells

    doi: 10.4049/jimmunol.1801227

    Figure Lengend Snippet: We propose that in addition to its direct antimicrobial activity, AG-30/5C contributed to host defense via MC degranulation through MRGPRX2. AG-30/5C contributes to wound healing via its action on vascular endothelial cells keratinocyte proliferation and migration through MRGPRX3 and MRGPRX4.

    Article Snippet: MRGPRX2-Tango (Addgene #66440) and MRGPRX4-Tango (Addgene#66442) plasmids were gifts from Bryan Roth ( 32 ).

    Techniques: Activity Assay, Migration

    (A) Effects on cells stably expressing myc-B 2 Rs from either species (positive control, BK). (B) Effect on cells transiently expressing human MRGPRX2 (positive control, compound 48/80). Tracings, obtained via Fura-2 fluorescence, represent calcium mobilization, in fold of basal values to show the time course of effects elicited by receptor ligands (conventionally applied at time 15 s, arrows). The number of determinations is indicated between parentheses.

    Journal: PeerJ

    Article Title: Species-specific pharmacology of maximakinin, an amphibian homologue of bradykinin: putative prodrug activity at the human B 2 receptor and peptidase resistance in rats

    doi: 10.7717/peerj.2911

    Figure Lengend Snippet: (A) Effects on cells stably expressing myc-B 2 Rs from either species (positive control, BK). (B) Effect on cells transiently expressing human MRGPRX2 (positive control, compound 48/80). Tracings, obtained via Fura-2 fluorescence, represent calcium mobilization, in fold of basal values to show the time course of effects elicited by receptor ligands (conventionally applied at time 15 s, arrows). The number of determinations is indicated between parentheses.

    Article Snippet: The vector MRGPRX2-Tango (human receptor sequence; ) was a gift from Dr. Bryan Roth (Addgene plasmid # 66440).

    Techniques: Stable Transfection, Expressing, Positive Control, Fluorescence

    Table 1

    Journal: The Journal of Clinical Investigation

    Article Title: Mutations in 2 distinct genetic pathways result in cerebral cavernous malformations in mice

    doi: 10.1172/JCI44393

    Figure Lengend Snippet: Table 1

    Article Snippet: Rabbit polyclonal antibody against PDCD10 was from Proteintech Group.

    Techniques: