Journal: BMC Women's Health

Article Title: AlphaVBeta3 Integrin expression within uterine endometrium in unexplained infertility: a prospective cohort study

doi: 10.1186/s12905-017-0438-3

Figure Lengend Snippet: Immunostained slide with anti-avβ3 integrin, DAB chromogen, magnification x 200, score 0 in a case of unexplained infertility group

Article Snippet: After blocking with non-immune 4% goat serum incubated with tissues for 30 min, the primary antibody using monoclonal mouse IgG antibodies directed against human αvβ3 integrin (Human integrin alphaV beta3 MAb (clone 23C6)®, MAB3050, R&D systems, Minneapolis, USA) was added to these sections.

Techniques:

Journal: BMC Women's Health

Article Title: AlphaVBeta3 Integrin expression within uterine endometrium in unexplained infertility: a prospective cohort study

doi: 10.1186/s12905-017-0438-3

Figure Lengend Snippet: Immunostained slide with anti-avβ3 integrin, DAB chromogen, magnification x 200, score 3 in a case of fertility group. Reactivity is mainly in the galndular epithelium)

Article Snippet: After blocking with non-immune 4% goat serum incubated with tissues for 30 min, the primary antibody using monoclonal mouse IgG antibodies directed against human αvβ3 integrin (Human integrin alphaV beta3 MAb (clone 23C6)®, MAB3050, R&D systems, Minneapolis, USA) was added to these sections.

Techniques:

Journal: BMC Women's Health

Article Title: AlphaVBeta3 Integrin expression within uterine endometrium in unexplained infertility: a prospective cohort study

doi: 10.1186/s12905-017-0438-3

Figure Lengend Snippet: Immunostained slide with anti- vβ3 integrin, DAB chromogen, magnification x 40 (Low Power), score 3 in A case of fertility group. Reactivity could be seen in both Luminal Epithelium (LE) the Glandular Epithelium (GE)

Article Snippet: After blocking with non-immune 4% goat serum incubated with tissues for 30 min, the primary antibody using monoclonal mouse IgG antibodies directed against human αvβ3 integrin (Human integrin alphaV beta3 MAb (clone 23C6)®, MAB3050, R&D systems, Minneapolis, USA) was added to these sections.

Techniques:

Journal: BMC Women's Health

Article Title: AlphaVBeta3 Integrin expression within uterine endometrium in unexplained infertility: a prospective cohort study

doi: 10.1186/s12905-017-0438-3

Figure Lengend Snippet: Immunostained slide with anti- vβ3 integrin, DAB chromogen, magnification x 200 (High Power), score 3 in A case of fertility group. Reactivity could be seen Mainly in Luminal Epithelium (red arrow) rather than the Glandular Epithelium (red star)

Article Snippet: After blocking with non-immune 4% goat serum incubated with tissues for 30 min, the primary antibody using monoclonal mouse IgG antibodies directed against human αvβ3 integrin (Human integrin alphaV beta3 MAb (clone 23C6)®, MAB3050, R&D systems, Minneapolis, USA) was added to these sections.

Techniques:

Journal: BMC Women's Health

Article Title: AlphaVBeta3 Integrin expression within uterine endometrium in unexplained infertility: a prospective cohort study

doi: 10.1186/s12905-017-0438-3

Figure Lengend Snippet: Comparison of endometrial thickness, subendometrial Doppler resistance index, and ανβ3-integrin score in both study groups

Article Snippet: After blocking with non-immune 4% goat serum incubated with tissues for 30 min, the primary antibody using monoclonal mouse IgG antibodies directed against human αvβ3 integrin (Human integrin alphaV beta3 MAb (clone 23C6)®, MAB3050, R&D systems, Minneapolis, USA) was added to these sections.

Techniques: Comparison, Control

Journal: BMC Women's Health

Article Title: AlphaVBeta3 Integrin expression within uterine endometrium in unexplained infertility: a prospective cohort study

doi: 10.1186/s12905-017-0438-3

Figure Lengend Snippet: Receiver-operating characteristic (ROC) curve for the discrimination between patients with unexplained infertility and normal controls using a : Endometrial thickness, b : Subendometril RI, c : avβ3 integrin

Article Snippet: After blocking with non-immune 4% goat serum incubated with tissues for 30 min, the primary antibody using monoclonal mouse IgG antibodies directed against human αvβ3 integrin (Human integrin alphaV beta3 MAb (clone 23C6)®, MAB3050, R&D systems, Minneapolis, USA) was added to these sections.

Techniques:

Journal: BMC Women's Health

Article Title: AlphaVBeta3 Integrin expression within uterine endometrium in unexplained infertility: a prospective cohort study

doi: 10.1186/s12905-017-0438-3

Figure Lengend Snippet: Receiver-operating characteristic (ROC) curve analysis for the discrimination between patients with unexplained infertility and control group

Article Snippet: After blocking with non-immune 4% goat serum incubated with tissues for 30 min, the primary antibody using monoclonal mouse IgG antibodies directed against human αvβ3 integrin (Human integrin alphaV beta3 MAb (clone 23C6)®, MAB3050, R&D systems, Minneapolis, USA) was added to these sections.

Techniques: Control

Journal: BMC Women's Health

Article Title: AlphaVBeta3 Integrin expression within uterine endometrium in unexplained infertility: a prospective cohort study

doi: 10.1186/s12905-017-0438-3

Figure Lengend Snippet: Summary of studies looked at endometrial αVβ3 integrin in luteal phase of infertile women

Article Snippet: After blocking with non-immune 4% goat serum incubated with tissues for 30 min, the primary antibody using monoclonal mouse IgG antibodies directed against human αvβ3 integrin (Human integrin alphaV beta3 MAb (clone 23C6)®, MAB3050, R&D systems, Minneapolis, USA) was added to these sections.

Techniques: Control, Expressing

Journal: Bioactive Materials

Article Title: Phosphorylation inhibition of protein-tyrosine phosphatase 1B tyrosine-152 induces bone regeneration coupled with angiogenesis for bone tissue engineering

doi: 10.1016/j.bioactmat.2020.12.025

Figure Lengend Snippet: 152RM induces the migration of MSCs partly through the FAK/STAT3 signaling pathway. (A) Transwell assays for the migration of MSCs using 152RM (n = 5 each). Representative crystal violet staining images are shown in the left panel. Quantification of cell migration is shown in the right panel. Scale bar, 100 μm. (B) RNA-seq analysis showed the alteration of cell migration-specific gene expression in MSCs cultured with 152RM (n = 3 each). (C) Relative mRNA expression levels of cell migration-specific genes in MSCs cultured with 152RM (n = 5 each). (D) Quantification of the transwell assay and cell wound scratch assay after culture with 152RM, a CXCR4 inhibitor (AMD3100) and an Integrin αvβ3 inhibitor (Cyclo(-RGDfK)) (n = 5 each). (E) Western blot analysis of the expression of E-cadherin in MSCs after the addition of 152RM (n = 5 per group). (F) Gene set enrichment analysis (GSEA) plots showing upregulation of the JAK/STAT signaling pathway in MSCs cultured with 152RM (n = 3 each). (G) RNA-seq analysis showed alterations in JAK/STAT signaling pathway-related gene expression in MSCs cultured with 152RM (n = 3 each). (H) Western blot analysis of the expression of CXCR4, integrin αvβ3, p-Jak2, Jak2, p-FAK, FAK, p-STAT3 and STAT3 in MSCs (pretreated with a CXCR4 inhibitor (AMD3100) and an integrin αvβ3 inhibitor (cyclo(-RGDfK))) after the addition of 152RM (n = 5 per group). Data are shown as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001; ANOVA and Student's t -test were employed. For all panels in this figure, data are representative of three independent experiments.

Article Snippet: Briefly, the bone sections were incubated with individual primary antibodies against mouse CD31 (ab28364; Abcam), endomucin (V.7C7; Santa Cruz), Ki67 (AF7617; R&D), beta-catenin (8480, CST), osterix (bs-1110R; Bioss), osteocalcin (bs-0470R; Bioss), Runx2 (bs-1134R; Bioss), DLL4 (bs-6044R; Bioss), Notch1 (bs-1335R; Bioss), Noggin (bs-2975R; Bioss), CXCR4 D1S7W; Cell Signaling Technology), integrin αvβ3 (bs-1310R; Bioss), CD90 (bs-20640R; Bioss), CD105 (bs-0579R; Bioss), CD271 (bs-0161R; Bioss), OPN (bs-23258R; Bioss), and phospho-VEGFR2(bs-2674R; Bioss) overnight at 4 °C.

Techniques: Migration, Staining, RNA Sequencing Assay, Expressing, Cell Culture, Transwell Assay, Wound Healing Assay, Western Blot

Journal: Bioactive Materials

Article Title: Phosphorylation inhibition of protein-tyrosine phosphatase 1B tyrosine-152 induces bone regeneration coupled with angiogenesis for bone tissue engineering

doi: 10.1016/j.bioactmat.2020.12.025

Figure Lengend Snippet: 152RM induces ECs migration partly through the FAK/ERK signaling pathway. (A) Transwell assay for the migration of ECs using 152RM (n = 5 each). Scale bar, 100 μm. Quantification of cell migration was performed (right). (B) Relative mRNA expression levels of cell migration-specific genes in ECs cultured with 152RM (n = 5 each). (C) Representative immunostaining images of p-VEGFR2 (red) ECs with or without 152RM (n = 5 per group). Scale bar, 100 μm. (D) Quantification of transwell assays after culture with 152RM, a VEGFR2 inhibitor (Ki8751) and an integrin αvβ3 inhibitor cyclo(-RGDfK) (n = 5 each). (E) Western blot analysis of the expression of integrin αvβ3, p-VEGFR2, p-FAK, FAK, p-ERK1/2 and ERK1/2 in ECs (pretreated with a VEGFR2 inhibitor (Ki8751) and an integrin αvβ3 inhibitor (cyclo(-RGDfK))) after the addition of 152RM (n = 5 per group). (F) Schematic illustration of the role of 152RM in promoting ECs migration. Data are shown as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001; ANOVA and Student's t -test were employed. For all panels in this figure, data are representative of three independent experiments.

Article Snippet: Briefly, the bone sections were incubated with individual primary antibodies against mouse CD31 (ab28364; Abcam), endomucin (V.7C7; Santa Cruz), Ki67 (AF7617; R&D), beta-catenin (8480, CST), osterix (bs-1110R; Bioss), osteocalcin (bs-0470R; Bioss), Runx2 (bs-1134R; Bioss), DLL4 (bs-6044R; Bioss), Notch1 (bs-1335R; Bioss), Noggin (bs-2975R; Bioss), CXCR4 D1S7W; Cell Signaling Technology), integrin αvβ3 (bs-1310R; Bioss), CD90 (bs-20640R; Bioss), CD105 (bs-0579R; Bioss), CD271 (bs-0161R; Bioss), OPN (bs-23258R; Bioss), and phospho-VEGFR2(bs-2674R; Bioss) overnight at 4 °C.

Techniques: Migration, Transwell Assay, Expressing, Cell Culture, Immunostaining, Western Blot

Journal: Bioactive Materials

Article Title: Phosphorylation inhibition of protein-tyrosine phosphatase 1B tyrosine-152 induces bone regeneration coupled with angiogenesis for bone tissue engineering

doi: 10.1016/j.bioactmat.2020.12.025

Figure Lengend Snippet: DBM-MSN/152RM scaffolds coordinate the recruitment of MSCs and ECs in vivo . (A) HE staining images demonstrating the recruitment of MSCs and ECs 1 week after DBM, DBM-MSN, DBM/152RM and DBM-MSN/152RM scaffold implantation (n = 5 rats per group). Scale bar, 100 μm. (B) Co-immunofluorescence staining of CXCR4, integrin αvβ3 and CD271 in MSCs from 1 week after DBM, DBM-MSN, DBM/152RM and DBM-MSN/152RM scaffold implantation (n = 5 rats per group). Scale bar, 100 μm. (C) Immunofluorescence staining of CD90 + CD105 + MSCs from 1 week after DBM, DBM-MSN, DBM/152RM and DBM-MSN/152RM scaffold implantation (n = 5 rats per group). Scale bar, 100 μm. (D) Co-immunofluorescence staining of emcn and integrin αvβ3 in type H vessels from 4 weeks after DBM, DBM-MSN, DBM/152RM and DBM-MSN/152RM scaffold implantation (n = 5 rats per group). Scale bar, 100 μm. (E) Immunofluorescence staining of CD31 + in EPCs from 4 weeks after DBM, DBM-MSN, DBM/152RM and DBM-MSN/152RM scaffold implantation (n = 5 rats per group). Scale bar, 100 μm. Data are shown as the mean ± SD. ns P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001; ANOVA was employed. For all panels in this figure, data are representative of three independent experiments.

Article Snippet: Briefly, the bone sections were incubated with individual primary antibodies against mouse CD31 (ab28364; Abcam), endomucin (V.7C7; Santa Cruz), Ki67 (AF7617; R&D), beta-catenin (8480, CST), osterix (bs-1110R; Bioss), osteocalcin (bs-0470R; Bioss), Runx2 (bs-1134R; Bioss), DLL4 (bs-6044R; Bioss), Notch1 (bs-1335R; Bioss), Noggin (bs-2975R; Bioss), CXCR4 D1S7W; Cell Signaling Technology), integrin αvβ3 (bs-1310R; Bioss), CD90 (bs-20640R; Bioss), CD105 (bs-0579R; Bioss), CD271 (bs-0161R; Bioss), OPN (bs-23258R; Bioss), and phospho-VEGFR2(bs-2674R; Bioss) overnight at 4 °C.

Techniques: In Vivo, Staining, Immunofluorescence

Journal: Cell Adhesion & Migration

Article Title: Selective binding and lateral clustering of α 5 β 1 and α v β 3 integrins: Unraveling the spatial requirements for cell spreading and focal adhesion assembly

doi: 10.1080/19336918.2016.1163453

Figure Lengend Snippet: Cell spreading kinetics on nanopatterned surfaces functionalized with integrin selective ligands. (A) Progression of projected cell area during spreading on nanopatterned surfaces with interparticle distances of 30, 60, or 90 nm, and functionalized with α5β1 (white) and αvβ3 (black) integrin selective ligands. (B) Maximum projected cell area on the different surfaces. Error bars indicate SEM of 3 independent repeats.

Article Snippet: Cells were then washed with warm PBS, and fixed in 3.7% paraformaldehyde in PBS for 20 min. Post-fixation, cells were permeabilized with 0.1% TritonX-100 in PBS for 5 min, blocked with 1% bovine serum albumin (BSA) in PBS, and incubated for 1 hr with the following antibodies: mouse anti-human αvβ3 integrin (Millipore, MAB1976), rat anti-human α5-integrin (MABII, kindly provided by K. Yamada), mouse anti- human vinculin (Sigma, V9131), and rabbit anti-human zyxin (Synaptic Systems, 307011).

Techniques:

Journal: Cell Adhesion & Migration

Article Title: Selective binding and lateral clustering of α 5 β 1 and α v β 3 integrins: Unraveling the spatial requirements for cell spreading and focal adhesion assembly

doi: 10.1080/19336918.2016.1163453

Figure Lengend Snippet: Focal adhesions in cells adhering to nanopatterned surfaces functionalized with integrin α5β1 and αvβ3 integrin selective ligands. (A) Indirect immunofluorescence staining of vinculin (green), phosphorylated paxillin (red), and actin (blue) in U2OS cells. Insets are a magnification of separate stainings for vinculin and phosphorylated paxillin, in the cell region delineated by the white box. Cells adhering for 4 hr to α5β1 (first row) and αvβ3 integrin selective ligands (second row) at spacings of 30 nm (left), 60 nm (middle), and 90 nm (right) were imaged by wide-field microscopy. (B) Analysis of vinculin cluster size; and (C) Analysis of phosphorylated paxillin (PY118) cluster size in U2OS cells. Box plots indicate cluster area values between 25% and 75%, and whiskers between 10% and 90% of the data range. The line in the box plot indicates the median value. p * < 0.001.

Article Snippet: Cells were then washed with warm PBS, and fixed in 3.7% paraformaldehyde in PBS for 20 min. Post-fixation, cells were permeabilized with 0.1% TritonX-100 in PBS for 5 min, blocked with 1% bovine serum albumin (BSA) in PBS, and incubated for 1 hr with the following antibodies: mouse anti-human αvβ3 integrin (Millipore, MAB1976), rat anti-human α5-integrin (MABII, kindly provided by K. Yamada), mouse anti- human vinculin (Sigma, V9131), and rabbit anti-human zyxin (Synaptic Systems, 307011).

Techniques: Immunofluorescence, Staining, Microscopy

Journal: Cell Adhesion & Migration

Article Title: Selective binding and lateral clustering of α 5 β 1 and α v β 3 integrins: Unraveling the spatial requirements for cell spreading and focal adhesion assembly

doi: 10.1080/19336918.2016.1163453

Figure Lengend Snippet: α5 and αvβ3 clusters in U2OS cells adhering to nanopatterned surfaces functionalized with α5β1 and αvβ3 integrin selective ligands. (A) Cells adhering to surfaces with 30 nm interparticle spacing; and (B) Cells adhering to surfaces with 60 nm particle spacing. Upper row: Cells adhering to α5β1 integrin selective ligands. Lower row: Cells adhering to αvβ3 integrin selective ligands. Left: Staining for α5 clusters. Middle: Staining for αvβ3 clusters. Right: Lookup table displaying the colocalization of α5 and αvβ3 integrin clusters (pixel with positive signals for both integrins are shown in yellow).

Article Snippet: Cells were then washed with warm PBS, and fixed in 3.7% paraformaldehyde in PBS for 20 min. Post-fixation, cells were permeabilized with 0.1% TritonX-100 in PBS for 5 min, blocked with 1% bovine serum albumin (BSA) in PBS, and incubated for 1 hr with the following antibodies: mouse anti-human αvβ3 integrin (Millipore, MAB1976), rat anti-human α5-integrin (MABII, kindly provided by K. Yamada), mouse anti- human vinculin (Sigma, V9131), and rabbit anti-human zyxin (Synaptic Systems, 307011).

Techniques: Staining

Journal: Cell Adhesion & Migration

Article Title: Selective binding and lateral clustering of α 5 β 1 and α v β 3 integrins: Unraveling the spatial requirements for cell spreading and focal adhesion assembly

doi: 10.1080/19336918.2016.1163453

Figure Lengend Snippet: α5β1 and αvβ3 integrin blocking. (A) Phase contrast micrographs of U2OS cells incubated with α5β1 and αvβ3 integrin selective ligands, and seeded on nanopatterned surfaces functionalized with these ligands. Upper row: Cells adhering to α5β1integrin selective ligands. Lower row: Cells adhering to αvβ3 integrin selective ligands. Left: No integrin blocking. Middle: α5β1 integrin blocking. Right: αvβ3 integrin blocking. (B) Indirect immunofluorescence staining of α5 (green) and αvβ3 clusters (red) in U2OS cells pre-incubated with the integrin selective ligands. Cells were seen to adhere to nanopatterned surfaces functionalized with α5β1 (upper row) and αvβ3 integrin selective ligands (lower row).

Article Snippet: Cells were then washed with warm PBS, and fixed in 3.7% paraformaldehyde in PBS for 20 min. Post-fixation, cells were permeabilized with 0.1% TritonX-100 in PBS for 5 min, blocked with 1% bovine serum albumin (BSA) in PBS, and incubated for 1 hr with the following antibodies: mouse anti-human αvβ3 integrin (Millipore, MAB1976), rat anti-human α5-integrin (MABII, kindly provided by K. Yamada), mouse anti- human vinculin (Sigma, V9131), and rabbit anti-human zyxin (Synaptic Systems, 307011).

Techniques: Blocking Assay, Incubation, Immunofluorescence, Staining

Journal: Biology

Article Title: Engineering a Dual Specificity γδ T-Cell Receptor for Cancer Immunotherapy

doi: 10.3390/biology13030196

Figure Lengend Snippet: Evaluation of αvβ6-dependent activation of chimeric G115 γδ TCRs. ( A ) Analysis of integrin expression on A375 puro and A375-β6 cells. Red–integrin; blue–isotype control. Data are representative of 3 independent replicates. Co-cultures were performed between A375 puro ( B ) and A375-β6 ( C ) tumour cells and untransduced (untrans) or transduced T-cell populations at an effector-to-target ratio of 1:1 for 72 h. Residual tumour cell viability was determined using an MTT assay (mean ± SEM of indicated replicates). Statistical analysis was performed using one-way ANOVA; **** p < 0.0001. ( D ) Supernatants collected from co-cultures described in ( B , C ) were analysed for IFN-γ by ELISA (mean ± SEM; n = 21–45 from 4–6 independent donors). Statistical analysis was performed using one-way ANOVA; * p < 0.05; ** p < 0.01.

Article Snippet: Integrin expression was detected by flow cytometry using the following antibodies: αvβ3 integrin (FAB3050A, R&D Systems, Minneapolis, MN, USA), αvβ5 integrin (FAB2528A, R&D systems), αvβ6 integrin (FAB4155A, R&D Systems) and αvβ8 integrin (FAB4775A, R&D systems).

Techniques: Activation Assay, Expressing, Control, MTT Assay, Enzyme-linked Immunosorbent Assay

Journal: Biology

Article Title: Engineering a Dual Specificity γδ T-Cell Receptor for Cancer Immunotherapy

doi: 10.3390/biology13030196

Figure Lengend Snippet: Evaluation of PAg-dependent activation of chimeric G115 γδ TCRs by ffLuc-expressing K562 cells. ( A ) Analysis of αvβ6 integrin expression on ffLuc + K562 cells. Red–integrin; blue–isotype control. Data are representative of 3 independent replicates. ( B ) Firefly luciferase-expressing K562 cells were pre-incubated with the indicated Zol concentration for 24 h prior to the establishment of co-cultures with untrans(duced) or transduced T-cell populations at an effector to target ratio 1:1 for 72 h. Data show mean ± SEM of residual K562 viability (n = 6–11 from 4 independent donors), as determined by luciferase assay. Statistical analysis was performed using two-way ANOVA; *** p < 0.001; **** p < 0.0001. ( C ) Supernatants collected from co-cultures described in B were analysed for IFN-γ by ELISA (mean ± SEM; n = 19 from 3 independent donors). Statistical analysis was performed using two-way ANOVA; *** p < 0.001; **** p < 0.0001.

Article Snippet: Integrin expression was detected by flow cytometry using the following antibodies: αvβ3 integrin (FAB3050A, R&D Systems, Minneapolis, MN, USA), αvβ5 integrin (FAB2528A, R&D systems), αvβ6 integrin (FAB4155A, R&D Systems) and αvβ8 integrin (FAB4775A, R&D systems).

Techniques: Activation Assay, Expressing, Control, Luciferase, Incubation, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Biology

Article Title: Engineering a Dual Specificity γδ T-Cell Receptor for Cancer Immunotherapy

doi: 10.3390/biology13030196

Figure Lengend Snippet: Evaluation of the anti-tumour activity of chimeric G115 γδ TCRs against BxPC3 pancreatic tumour cells. ( A ) Analysis of integrin expression on BxPC3 cells. Red–integrin; blue–isotype control. Data are representative of 3 independent replicates. Co-cultures were performed between BxPC3 tumour cells (No Zol; B ) or Zol-sensitised BxPC3 tumour cells (+Zol; C ) and untrans(duced) or transduced T-cell populations at an effector to target ratio of 1:1 for 72 h. Tumour cell viability was determined using an MTT assay (mean ± SEM of indicated replicates). Statistical analysis was performed using one-way ANOVA; **** p < 0.0001, * p < 0.05, N/S–not significant.

Article Snippet: Integrin expression was detected by flow cytometry using the following antibodies: αvβ3 integrin (FAB3050A, R&D Systems, Minneapolis, MN, USA), αvβ5 integrin (FAB2528A, R&D systems), αvβ6 integrin (FAB4155A, R&D Systems) and αvβ8 integrin (FAB4775A, R&D systems).

Techniques: Activity Assay, Expressing, Control, MTT Assay

Journal: Biology

Article Title: Engineering a Dual Specificity γδ T-Cell Receptor for Cancer Immunotherapy

doi: 10.3390/biology13030196

Figure Lengend Snippet: Evaluation of the anti-tumour activity of chimeric G115 γδ TCRs against Panc1 pancreatic tumour cells. ( A ) Analysis of integrin expression on Panc1 cells. Red–integrin; blue–isotype control. Data are representative of 3 independent replicates. Co-cultures were performed between Panc1 tumour cells (No Zol; B ) or Zol-sensitised Panc1 tumour cells (+ Zol; C ) and untransduced (untrans) or transduced T-cell populations at an effector to target ratio of 1:1 for 72 h. Tumour cell viability was determined using an MTT assay (mean ± SEM of indicated replicates). Statistical analysis was performed using one-way ANOVA; **** p < 0.0001, ** p < 0.01, N/S–not significant.

Article Snippet: Integrin expression was detected by flow cytometry using the following antibodies: αvβ3 integrin (FAB3050A, R&D Systems, Minneapolis, MN, USA), αvβ5 integrin (FAB2528A, R&D systems), αvβ6 integrin (FAB4155A, R&D Systems) and αvβ8 integrin (FAB4775A, R&D systems).

Techniques: Activity Assay, Expressing, Control, MTT Assay

Journal: Tissue Engineering. Part A

Article Title: Exogenous MC3T3 Preosteoblasts Migrate Systemically and Mitigate the Adverse Effects of Wear Particles

doi: 10.1089/ten.tea.2012.0086

Figure Lengend Snippet: Immunohistochemical staining of a particle-infused femur (left block) and a saline-infused femur (right block). All sections were obtained from the diaphyseal cortex. DAPI identified all cells (A), antihuman vitronectin receptor αVβ3 identified osteoclasts (B), antiosteocalcin identified osteoblasts (C), and overlaying all three identified any costaining (D). Osteoclasts reside along the trabecular bone and the endosteal cortices (representative areas denoted by *), predominantly so in the case of saline-infused femora. The experimental femora had a much greater extent of osteoblast staining (representative areas indicated with #). All images are 200×magnification. Color images available online at www.liebertpub.com/tea

Article Snippet: In the second staining protocol, the mouse anti-human vitronectin receptor αVβ3 and the rabbit anti-mouse osteocalcin (both Santa Cruz Biotechnology) tagged osteoclasts and osteoblasts, respectively.

Techniques: Immunohistochemical staining, Staining, Blocking Assay, Saline

Journal: PLoS ONE

Article Title: Endocytic Mechanism of Internalization of Dietary Peptide Lunasin into Macrophages in Inflammatory Condition Associated with Cardiovascular Disease

doi: 10.1371/journal.pone.0072115

Figure Lengend Snippet: Two-dimensional optical planes demonstrate the immunocytochemical localization of lunasin (red), αVβ3 integrin (green) and nuclei (blue) ( Figure 1A ). Unstimulated macrophages or macrophages have been induced with LPS (10 nM), vitronectin (130 nM) or the two molecules in combination for 24 h and treated with lunasin (1 µM) for 1 h. Compared to the PBS control ( A–B ), lunasin uptake increased sequentially upon stimulation with LPS ( C–D ), vitronectin ( E–F ), or both molecules in combination ( G–H ). Lunasin (1 µM) treatment reduced the basal expression level of αVβ3 integrin at the cellular membrane ( I–J ). The upregulation of the total basal expression level of αVβ3 integrin at the membrane in the presence of LPS ( K–L ), vitronectin ( M–N ) or both molecules in combination ( O–P ) was subsequently attenuated by lunasin treatment (1 µM). The merged image (with nuclei) shows increased lunasin uptake and a concomitant reduction in the level of αVβ3 integrin expression at the surface of the plasma membrane ( Q–X ), as well as their respective expression levels ( Figure 1B, 1C ). Four independent fields of view from three independent cellular replicates were merged together per treatment group. In , the data represent the average of triplicates or quadruplicates ± SE. Means with different letters are significantly different from each other (n = 3, p<0.05). US = unstimulated, PBS = phosphate-buffered saline, LPS = lipopolysaccharide (10 nM), VTN = vitronectin (130 nM), LUN = lunasin (1 µM). Bar = 10 µm.

Article Snippet: The mouse antibody to αVβ3 integrin was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Control, Expressing, Membrane, Clinical Proteomics, Saline

Journal: PLoS ONE

Article Title: Endocytic Mechanism of Internalization of Dietary Peptide Lunasin into Macrophages in Inflammatory Condition Associated with Cardiovascular Disease

doi: 10.1371/journal.pone.0072115

Figure Lengend Snippet: Two-dimensional optical planes demonstrating immunocytochemical localization of lunasin (red), αVβ3 integrin (green) and nuclei (blue) ( Figure 5A ). Macrophages were pretreated with BFA (71 µM) for up to 6 h. BFA pretreatment inhibited lunasin internalization over all of the time points ( B–D ). Compared to the PBS control ( E ), lunasin treatment (1 µM) reduced the expression of αVβ3 integrin at the membrane after 30 min of BFA pretreatment ( F ), but αVβ3 integrin expression remained unaffected at all other time points ( G–H ). Merged image (with nuclei) showing the effect of lunasin uptake and αVβ3 integrin expression on BFA pretreatment ( I–L ), and their respective quantified expressions ( Figure 5B, 5C ). In , data represent the average of triplicates or quadruplicates ± SE. Four independent fields of view from three independent replicates of cells were merged together per treatment group. Means with different letters are significantly different from each other (n = 3, p<0.05). Bar = 10 µm.

Article Snippet: The mouse antibody to αVβ3 integrin was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Control, Expressing, Membrane

Journal: PLoS ONE

Article Title: Endocytic Mechanism of Internalization of Dietary Peptide Lunasin into Macrophages in Inflammatory Condition Associated with Cardiovascular Disease

doi: 10.1371/journal.pone.0072115

Figure Lengend Snippet: Two-dimensional immunocytochemical planes show lunasin (red), αVβ3 integrin, caveolin-1 or clathrin (green) and nuclei (blue) ( Figure 6A ). Macrophages were pretreated for 3 min with their respective endocytic inhibitors. Expression of αVβ3 integrin, caveolin-1 or clathrin ( E–H ) after treatment with lunasin and their specific inhibitors, respectively. Compared to the control ( A, I ), pretreatment with nystatin (54 µM, caveolae-mediated endocytic inhibitor) reduced lunasin internalization ( B, J ), while treatment with amantadine (1 mM, clathrin-mediated endocytic inhibitor) and amiloride (1 mM, macropinocytosis inhibitor) abolished lunasin uptake by macrophages ( C, J and D, L ). The effect of inhibitors on the endocytosis of lunasin . In , the data represent the average of triplicates or quadruplicates ± SE. Four independent fields of view from three independent replicates of cells were merged together per treatment group. Means with different letters are significantly different from each other (n = 3, p<0.05). Bar = 10 µm.

Article Snippet: The mouse antibody to αVβ3 integrin was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Expressing, Control

Journal: PLoS ONE

Article Title: Endocytic Mechanism of Internalization of Dietary Peptide Lunasin into Macrophages in Inflammatory Condition Associated with Cardiovascular Disease

doi: 10.1371/journal.pone.0072115

Figure Lengend Snippet: Macrophages imaged with live-cell scanning confocal microscopy for intracellular free Ca 2+ . The median optical planes of individual cells were monitored for up to 400 frames (6.67 min) for the levels of free intracellular Ca 2+ upon lunasin treatment ( Figure 7A ). Induction with LPS (10 nM) increased the levels of free intracellular Ca 2+ in the macrophages compared to the unstimulated control, and lunasin transiently reduced this effect. After 30 min, resurgence in the levels of free intracellular Ca 2+ was observed in the lunasin-treated cells. Echistatin, an αVβ3 inhibitor, prevented the modulation of the free intracellular Ca 2+ by lunasin. Quantification of the intensity demonstrates the effect of lunasin on the intracellular Ca 2+ levels over time ( Figure 7B ). Three independent fields of view from 10 independent replicates of cells were taken. Means within each treatment with different letters are significantly different from each other. An asterisk indicates that a treatment was significantly different from the media control (n = 3, p<0.05).

Article Snippet: The mouse antibody to αVβ3 integrin was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Confocal Microscopy, Control

Journal: FEBS letters

Article Title: Integrins and heparan sulfate proteoglycans on hepatic stellate cells (HSC) are novel receptors for HSC-derived exosomes

doi: 10.1002/1873-3468.12448

Figure Lengend Snippet: Primers used for RT-PCR

Article Snippet: In some binding experiments, HSC were pre-treated or co-incubated with 0-100 μg/ml RGD or RGE tripeptides (American Peptide, Sunnyvale CA), 0-100 μM EDTA (Sigma-Aldrich), 0-10 μM sodium chlorate (Sigma-Aldrich), 0-10 μM sodium sulfate (Sigma-Aldrich), 0-10 μg/ml rabbit anti-mouse integrin αvβ3 IgG (Bioss Inc, Woburn, MA), or 0-20 μg/ml rat anti-mouse integrin α5β1 IgG (Millipore, Temecula, CA) or 0-10 μg/ml rat anti-mouse integrin αM, (CD11b; Novus Biologicals Littleton CO).

Techniques:

Journal: FEBS letters

Article Title: Integrins and heparan sulfate proteoglycans on hepatic stellate cells (HSC) are novel receptors for HSC-derived exosomes

doi: 10.1002/1873-3468.12448

Figure Lengend Snippet: (A) PKH26-stained HSC-derived exosomes (red) were incubated with primary mouse HSC for 24hrs in the presence of RGD or RGE (0-100 μg/ml) in the incubation medium. Cells were analyzed by confocal microscopy (20X) (upper, showing a representative experiment) or measured for cell-associated fluorescence by spectrophotometry of cell lysates (lower; n = 5, *p < 0.001 vs. 0 μg/ml RGD, student’s t test). (B) Mouse HSC were pre-incubated with RGD or RGE (0-100 μg/ml) for 1 hr and excess peptide was removed by extensive washing prior to addition of PKH26-stained HSC exosomes (red) for 24hrs. The cells were analyzed by confocal microscopy (20X) (upper; showing a representative experiment) or by fluorescence intensity of cell lysates (lower; n = 5, *p < 0.001 vs. 0 μg/ml RGD, student’s t test. (C) PKH26-stained exosomes (red) were incubated with primary mouse HSC (green) for 24hrs with 0-100 μM EDTA. Cells were analyzed by confocal microscopy (20X) (left; showing a representative experiment) or fluorescence in cell lysates was determined spectrophotometrically (right; n = 5, *p < 0.001 vs. 0 μM EDTA, student’s t test). (D) HSC were transfected for 24 hrs with integrin αv or β1 siRNA and analyzed for expression of αv or β1 mRNA by RT-PCR (left; n = 9, * p < 0.001 vs. Ctrl, student’s t test) or levels of their corresponding proteins by Western blot using anti-integrin αvβ3 (center) or anti-integrin α5β1 antibodies (right) for which β-actin was used a loading control (n = 9, *p <0.001 vs. Ctrl, student’s t test). “Ctrl” represents cells treated with a scrambled siRNA sequence. (E) Mouse HSC were transfected for 24 hrs with siRNA to the integrin αv or β1 subunits, either individually or together. Cells were then stained with PKH-67 (green) for 1 hr and incubated with PKH26-stained exosomes (red) for 24 hrs. Blue, DAPI. Data are representative of 3 experiments. (F) Adhesion of mouse HSC or macrophages to a CCN2 substrate after pre-incubation of the cells with neutralizing anti-integrin αvβ3 (10 μg/ml), anti-integrin α5β1 (20 μg/ml), anti-integrin αM (10 μg/ml), or their non-immune IgG counterparts (at the same respective dose). n = 5, *p < 0.001 vs. Ctrl, student’s t test. (G) Mouse HSC were pre-incubated with neutralizing anti-integrin αvβ3, α5β1 or αM IgG or non-immune IgG for 1 hr prior to addition for 24 hrs of PKH26-stained HSC exosomes. Cells were analyzed by confocal microscopy (20X) (upper; showing a representative experiment) or by spectrophotometric quantification of fluorescence in cell lysates (lower; n = 6, *p < 0.001. +p < 0.05 vs. ctrl, student’s t test). (H) Recipient HSC were transfected with a parental or CCN2 3’-UTR luciferase reporter vector for 24hrs prior to 1-hr incubation with RGD, IgG, anti-integrin αvβ3 or anti-integrin α5β1. Cells were then incubated for 24 hrs in the presence of miR-214-enriched exosomes as described [17]. Exosome-mediated suppression of luciferase activity was inhibited by RGD or anti-integrin αvβ3. n = 9, *p < 0.001 vs. ctrl, student’s t test. (I) Immunocytochemical detection of CCN2, αSMA or collagen α1 in activated HSC alone (“ctrl”) or after 36-hr incubation with exosomes from D1-3 HSC, in the presence or absence of anti-integrin αvβ3 or anti-integrin α5β1. Data are representative of 3 experiments.

Article Snippet: In some binding experiments, HSC were pre-treated or co-incubated with 0-100 μg/ml RGD or RGE tripeptides (American Peptide, Sunnyvale CA), 0-100 μM EDTA (Sigma-Aldrich), 0-10 μM sodium chlorate (Sigma-Aldrich), 0-10 μM sodium sulfate (Sigma-Aldrich), 0-10 μg/ml rabbit anti-mouse integrin αvβ3 IgG (Bioss Inc, Woburn, MA), or 0-20 μg/ml rat anti-mouse integrin α5β1 IgG (Millipore, Temecula, CA) or 0-10 μg/ml rat anti-mouse integrin αM, (CD11b; Novus Biologicals Littleton CO).

Techniques: Staining, Derivative Assay, Incubation, Confocal Microscopy, Fluorescence, Spectrophotometry, Transfection, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Sequencing, Luciferase, Plasmid Preparation, Activity Assay

Journal: Biomedicines

Article Title: Albumin Fusion at the N-Terminus or C-Terminus of HM-3 Leads to Improved Pharmacokinetics and Bioactivities

doi: 10.3390/biomedicines9091084

Figure Lengend Snippet: HM-3/HSA inhibited FAK/Src signaling pathways by binding with integrin αvβ3 and α5β1. ( A , B ) The integrin monoclonal antibody-blocking assay was performed to verify the interaction between HM-3/HSA and both integrin α5β1 and αvβ3. The high binding rate of HM-3/HSA increased with B16F10 cells through binding with integrin αvβ3 and α5β1 of the cell surface, but the adhesion number was significantly decreased when B16F10 cells were blocked with the integrin αvβ3 or α5β1 antibody. Control: cells treated with the PBS vehicle only. Scale bar = 100 μm. Data shown are means ± standard deviation from three independent experiments. ** p < 0.01, *** p < 0.001, and # p < 0.05. ( C ) HM-3-HSA affected the FAK/Src signaling pathway in B16F10 cells. The B16F10 cells were treated with 0.25 μM HSA-HM-3 or 0.25 μM HM-3-HSA for 48 h. GAPDH was used as the internal reference. Data shown are means ± standard deviation from two independent experiments.

Article Snippet: For the adhesion inhibiting assay, cell suspensions were incubated with the anti-α5β1 monoclonal antibody (Bioss bs-1310R; 1:500) or anti-αvβ3 monoclonal antibody (Sino Biological CT014-MM01, Beijing, China; 1:500) for 1 h at RT.

Techniques: Binding Assay, Antibody Blocking Assay, Standard Deviation