Journal: EBioMedicine

Article Title: Proapoptotic Cyclic Peptide BC71 Targets Cell-Surface GRP78 and Functions as an Anticancer Therapeutic in Mice

doi: 10.1016/j.ebiom.2018.06.004

Figure Lengend Snippet: BC71 targets cell-surface GRP78 but not αvβ5 integrin to induce apoptosis. (a) BC71 induces HUVECs apoptosis in a dose-dependent manner. HUVECs were treated with BC71 (concentration range: 12.5, 25, 50, 100 μM) for 24 h and apoptosis was determined using the cell death ELISA kit (Roche). (b) Anti-GRP78 N-terminal domain antibody blocked the apoptosis function of BC71 in a dose-dependent manner. The apoptosis of the combined treatment with increasing amount of anti-GRP78 N-terminal domain antibody and 100 μM BC71 for 24 h was measured using the Cell Death Detection ELISA. (c) Anti-GRP78 C-terminal domain antibody and (d) anti-αvβ5 antibody did not block BC71 induced apoptosis. For clarity of presentation, data were normalized with that of non-treated (VEGF only) cells, which was set as 1. Data are expressed as mean ± standard error of the mean. The results are representative of at least three independent experiments. Statistical significance was determined using ANOVA. *P < 0.05; **P < 0.01, n ≥ 3.

Article Snippet: Antibodies against integrin αvβ5 (P1F76; Santa Cruz Biotechnology, Santa Cruz, CA, USA), GRP78 (A-10, Santa Cruz Biotechnology) and GRP78 (C-20, Santa Cruz Biotechnology) were used for neutralization.

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

Journal: International journal of molecular sciences

Article Title: Treadmill Exercise Alleviates Cognition Disorder by Activating the FNDC5: Dual Role of Integrin αV/β5 in Parkinson's Disease.

doi: 10.3390/ijms24097830

Figure Lengend Snippet: Figure 7. Effect of treadmill exercise on FNDC5/integrinαVβ5-mediated BDNF levels in MPTP- induced chronic PD model and verification of FNDC5–integrin αV β5 interactions. (A) Protein levels of FNDC5, integrin αV, integrin β5 and BDNF were examined with Western blotting. (B–E) Quan- tification of FNDC5 (F(2,6) = 13.59, p = 0.006), integrin αV (F(2,6) = 58.63, p < 0.001), integrin β5 (F(2,6) = 27.36, p = 0.001), and BDNF (F(2,6) = 33.06, p < 0.001) levels (n = 3). (F,G) Representative immunofluorescence images and summarized data of staining for integrin (t(10) = 33.18) in HT22 (hippocampal neuron cell line) cells. Scale bar = 100 µm, n = 6. (H,I) Interactions between FNDC5 and integrin proteins (F(2,6) = 45.77, p < 0.001) were examined by Co-IP, n = 3. Data represent the mean ± SEM; * p < 0.05, ** p < 0.01 *** p < 0.001 vs. control, # p < 0.05 vs. MPTP group. M (MPTP), ME (MPTP + exercise), IP (immunoprecipitation), and IB (immunoblotting).

Article Snippet: These primary antibodies included TH (1:1000, ab112, Abcam, Shanghai, China), PSD95 (1:1000, 3450S, CST, Fall River, MA, USA), Synapsin (1:1000, NB300-104, NOVUS (Shanghai) Co. Ltd, Centennial, CO, USA), CAMKII(1:1000, 50049S, CST, Fall River, MA, USA), SNAP47 (1:1000, ab172609, Abcam, Shanghai, China), Synaptophysin (1:1000, ab52636, Abcam, Shanghai, China), BDNF (1:200, ANT-010, Alomone Labs, Jerusalem, Israel), FNDC5 (1:1000, bs-8486R, Bioss, Beijing, China), integrin αV (1:200, sc-9969, Santa Cruz, Dallas, TX, USA), integrin β5 (1:200, sc398214, Santa Cruz, Dallas, TX, USA), and β-actin (1:5000, 60008-1-Ig, Proteintech, Wuhan, China).

Techniques: Western Blot, Staining, Co-Immunoprecipitation Assay, Control, Immunoprecipitation

Journal: International journal of molecular sciences

Article Title: Treadmill Exercise Alleviates Cognition Disorder by Activating the FNDC5: Dual Role of Integrin αV/β5 in Parkinson's Disease.

doi: 10.3390/ijms24097830

Figure Lengend Snippet: Figure 8. Effect of treadmill-exercise-induced FNDC5 levels on dopaminergic synaptic connections from substantia nigra to hippocampus. (A,B) Schematic diagram of virus injection and expres- sion in SNpc and hippocampus. Scale bar = 500 µm (C,D) Representative DA neuron projection (F(2,15) = 895.2, p < 0.001) in hippocampus DG region of mice (scale bar = 100 µm, 200 µm. N = 6). The white arrows showed anterograde tracer virus across postsynaptic positive cells. (E,F) Representative image of TH-positive neurons and branches (F(2,15) = 223.3, p < 0.001) in SNpc region of mice (scale bar = 50 µm, n = 6). (G,H) Interactions between CD90 and integrin proteins in hippocampus were examined by IP (t(4) = 11.67, n = 3, p < 0.001). Data are presented as mean ± SEM; *** p < 0.001 vs. control, and ## p < 0.01, ### p < 0.001 vs. MPTP group. M (MPTP), ME (MPTP + exercise), ME+FNDC5 (MPTP + exercise + FNDC5), IP (immunoprecipitation), and IB (immunoblotting).

Article Snippet: These primary antibodies included TH (1:1000, ab112, Abcam, Shanghai, China), PSD95 (1:1000, 3450S, CST, Fall River, MA, USA), Synapsin (1:1000, NB300-104, NOVUS (Shanghai) Co. Ltd, Centennial, CO, USA), CAMKII(1:1000, 50049S, CST, Fall River, MA, USA), SNAP47 (1:1000, ab172609, Abcam, Shanghai, China), Synaptophysin (1:1000, ab52636, Abcam, Shanghai, China), BDNF (1:200, ANT-010, Alomone Labs, Jerusalem, Israel), FNDC5 (1:1000, bs-8486R, Bioss, Beijing, China), integrin αV (1:200, sc-9969, Santa Cruz, Dallas, TX, USA), integrin β5 (1:200, sc398214, Santa Cruz, Dallas, TX, USA), and β-actin (1:5000, 60008-1-Ig, Proteintech, Wuhan, China).

Techniques: Virus, Injection, Control, Immunoprecipitation, Western Blot

Journal: International journal of molecular sciences

Article Title: Treadmill Exercise Alleviates Cognition Disorder by Activating the FNDC5: Dual Role of Integrin αV/β5 in Parkinson's Disease.

doi: 10.3390/ijms24097830

Figure Lengend Snippet: Figure 9. Schematic diagram of consistent exercise in PD mice promoting the synthesis of FNDC5 in the muscle and its release into the blood. FNDC5 was also u-regulated in the brain, resulting in a protective effect on the hippocampus. Direct effects: FNDC5 activated integrin αV and β5 receptors on hippocampal neurons and promoted the activation of the CREB-mediated BDNF pathway. Indirect effects: FNDC5 promoted the interaction between the integrin receptors on the hippocampal neurons and the CD90 on the dopaminergic neurons to maintain the dopaminergic synaptic connection from the substantia nigra to the hippocampus.

Article Snippet: These primary antibodies included TH (1:1000, ab112, Abcam, Shanghai, China), PSD95 (1:1000, 3450S, CST, Fall River, MA, USA), Synapsin (1:1000, NB300-104, NOVUS (Shanghai) Co. Ltd, Centennial, CO, USA), CAMKII(1:1000, 50049S, CST, Fall River, MA, USA), SNAP47 (1:1000, ab172609, Abcam, Shanghai, China), Synaptophysin (1:1000, ab52636, Abcam, Shanghai, China), BDNF (1:200, ANT-010, Alomone Labs, Jerusalem, Israel), FNDC5 (1:1000, bs-8486R, Bioss, Beijing, China), integrin αV (1:200, sc-9969, Santa Cruz, Dallas, TX, USA), integrin β5 (1:200, sc398214, Santa Cruz, Dallas, TX, USA), and β-actin (1:5000, 60008-1-Ig, Proteintech, Wuhan, China).

Techniques: Activation Assay

Journal: bioRxiv

Article Title: Tumor-resident regulatory T cells in pancreatic cancer express the αvβ5 integrin as a targetable activation marker

doi: 10.1101/2023.05.24.542137

Figure Lengend Snippet: a Representative confocal images of αvβ5 integrin + CD4 + Foxp3 + T cells (left) and NRP-1 + CD4 + Foxp3 + T cells (right) in the PDAC tissue of KPC-derived orthotopic PDAC mice. Arrows indicate αvβ5 integrin + or NRP-1 + Tregs (magenta). Green, CD4; red, Foxp3; blue, DAPI. The boxed areas are magnified. Scale bars, 20 μm. b A representative flow cytometry analysis showing the proportion of CD8 + T cells, CD4 + CD25 neg T cells (non-Tregs), CD4 + CD25 + Tregs that are positive for αvβ5 integrin, NRP-1, or both in KPC-derived orthotopic PDAC tumors. c Bar diagrams that summarize the findings from ( b ) and Supplementary Fig. 10. n = 5 per group. d Representative confocal images of αvβ5 integrin + Foxp3 + CD4 + Tregs and NRP-1 + CD4 + Tregs in human PDAC and spleen. Magenta, αvβ5 integrin or NRP-1; green, CD4; red, Foxp3; blue, DAPI. Scale bars, 20 μm. e The number of αvβ5 integrin-positive and NRP-1-positive CD4 + Foxp3 + Tregs was counted under a confocal microscope and the % positivity was calculated. n = 3. Statistical analysis, one-way ANOVA ( c ) and Welch’s test ( e ); p = 0.0003 ( c, left, CD8 vs Tregs), p = 0.0169 ( c, left, Non-Tregs vs Tregs), p = 0.0002 ( c, left, Tregs vs Spleen Tregs), p = 0.0003 ( c, center, CD8 vs Tregs), p = 0.0123 ( c, center, Non-Tregs vs Tregs), p < 0.0001 ( c, center, Tregs vs Spleen Tregs), p = 0.0008 ( c, right, CD8 vs Tregs), p = 0.0109 ( c, right, Non-Tregs vs Tregs), p = 0.0019 ( c, right, Tregs vs Spleen Tregs), p = 0.0071 ( e, left), p = 0.4858 ( e, right). Error bars, mean ± standard error. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; N.S., not significant.

Article Snippet: After removing dead cells with a Dead Cell Removal kit (Miltenyi Biotec), the cells were treated with a PE-conjugated anti-CD25 Ab (Miltenyi Biotec), PE-conjugated anti-CCR8 Ab (eBioscience), and/or Alexa Fluor ® 647-conjugated anti-β5 integrin Ab (clone ALULA; BD Biosciences) combined with an anti-PE or -AF647 magnetic separation technique (Miltenyi Biotec) to enrich for CCR8 + iTregs, αvβ5 integrin + CCR8 + iTregs, or αvβ5 integrin neg CCR8 + iTregs.

Techniques: Derivative Assay, Flow Cytometry, Microscopy

Journal: bioRxiv

Article Title: Tumor-resident regulatory T cells in pancreatic cancer express the αvβ5 integrin as a targetable activation marker

doi: 10.1101/2023.05.24.542137

Figure Lengend Snippet: a-e CD4 + T cells isolated from the spleens of healthy C57B6129SF1/J hybrid mice were expanded for 3 days in vitro in the presence of KPC-derived PDAC cells. Flow cytometry was performed for subsequent analyses. ( a ) Expression of αvβ5 integrin and NRP-1 on CD4 + CD25 + Tregs expanded with or without the PDAC cells. n = 4 per group. ( b ) Expression of αvβ5 integrin and NRP-1 on CD4 + CD25 + Tregs and non-Treg CD4 + CD25 neg T cells expanded with the PDAC cells. n = 3 per group. ( c ) FAM-iRGD binding to the non-Tregs (blue line) and Tregs (red line) shown in ( b ). The bar diagram summarizes the median fluorescence intensity (MFI) from 4 independent experiments. ( d ) Dose-dependent inhibition of FAM-iRGD binding by an anti-αvβ5 integrin blocking Ab to Tregs that were expanded in the presence of PDAC cells. Values were normalized against isotype control. n = 3 per group. Statistical analysis was performed between the isotype control and anti-αvβ5 integrin values. ( e ) Tregs and non-Tregs were expanded in the presence of PDAC cells with or without iRGD. Apoptosis was quantified by measuring annexin V and 7-AAD double positive cells by flow cytometry. n = 3. f, g In vitro binding of FAM-iRGD to non-Treg CD4 + CD25 neg T cells (blue line) and CD4 + CD25 + Tregs (red line) isolated from the PDAC tissue ( f ) or the spleen ( g ) of KPC-derived orthotopic PDAC mice. The bar diagrams summarize the MFI from 4 independent experiments. h Representative confocal images of Foxp3 + T cells (red) in the PDAC tissue of KPC-derived orthotopic PDAC mice that received an intravenous injection of FAM-iRGD (green). Blue, DAPI. Arrows indicate Tregs positive for iRGD. The boxed area is magnified. Scale bars, 20 μm. Statistical analyses; Mann-Whitney U test ( a , c , f , g ), Welch’s t test ( b , e ), and one sample Wilcoxon signed rank test ( d ); p = 0.0286 ( a , top), p = 0.3429 ( a , bottom), p = 0.0058 ( b , top), p = 0.0086 ( b , bottom), p = 0.0017 ( c ), p = 0.7272 ( d , 0.3), p = 0.1766 ( d , 1.0), p = 0.0052 ( d , 3.0), p = 0.0087 ( e , top), p = 0.6583 ( e , bottom), p = 0.0286 ( f ), p = 0.6857 ( g ). Error bars, mean ± standard error; * p < 0.05; ** p < 0.01; N.S., not significant.

Article Snippet: After removing dead cells with a Dead Cell Removal kit (Miltenyi Biotec), the cells were treated with a PE-conjugated anti-CD25 Ab (Miltenyi Biotec), PE-conjugated anti-CCR8 Ab (eBioscience), and/or Alexa Fluor ® 647-conjugated anti-β5 integrin Ab (clone ALULA; BD Biosciences) combined with an anti-PE or -AF647 magnetic separation technique (Miltenyi Biotec) to enrich for CCR8 + iTregs, αvβ5 integrin + CCR8 + iTregs, or αvβ5 integrin neg CCR8 + iTregs.

Techniques: Isolation, In Vitro, Derivative Assay, Flow Cytometry, Expressing, Binding Assay, Fluorescence, Inhibition, Blocking Assay, Injection, MANN-WHITNEY

Journal: bioRxiv

Article Title: Tumor-resident regulatory T cells in pancreatic cancer express the αvβ5 integrin as a targetable activation marker

doi: 10.1101/2023.05.24.542137

Figure Lengend Snippet: Naïve CD4 + T cells were isolated from the spleens of healthy C57B6129SF1/J hybrid mice by magnetically removing CD4 neg T cells and CD25 + T cells. The pool enriched for naïve CD4 + T cells was cultured in vitro with anti-CD3/CD28 Abs in the presence or absence of TGF-β1 for 3 days, and analyzed for αvβ5 integrin and NRP-1 expression by flow cytometry. ( Top row) Naïve CD4 + T cells (blue box) enriched from mouse splenocytes. A minor population of CD4 + Foxp3 + T cells was present (red box). ( Middle row ) Treating the pool in the top row with anti-CD3/CD28 Abs and TGF-β1 yielded approximately 40% of CD4 + Foxp3 + T cells (red box) and 56% of CD4 + Foxp3 neg T cells (blue box). ( Bottom row ) Treating the pool in the top row with anti-CD3/CD28 Abs alone did not change the proportion of the CD4 + T cells. Nearly 95% of the cells remained negative for Foxp3 (blue box). Representative dot plots showing the proportion of CD4 + Foxp3 + T cells (left panels) and the expression of αvβ5 integrin and NRP-1 on the indicated population are presented. The bar diagrams summarize the proportion of αvβ5 + cells in the indicated population. n = 3 per study. Statistical analysis, Welch’s t test; p = 0.4115 (top), p = 0.0013 (middle), p = 0.0032 (bottom). Error bars, mean ± standard error; ** p < 0.01; N.S., not significant.

Article Snippet: After removing dead cells with a Dead Cell Removal kit (Miltenyi Biotec), the cells were treated with a PE-conjugated anti-CD25 Ab (Miltenyi Biotec), PE-conjugated anti-CCR8 Ab (eBioscience), and/or Alexa Fluor ® 647-conjugated anti-β5 integrin Ab (clone ALULA; BD Biosciences) combined with an anti-PE or -AF647 magnetic separation technique (Miltenyi Biotec) to enrich for CCR8 + iTregs, αvβ5 integrin + CCR8 + iTregs, or αvβ5 integrin neg CCR8 + iTregs.

Techniques: Isolation, Cell Culture, In Vitro, Expressing, Flow Cytometry

Journal: bioRxiv

Article Title: Tumor-resident regulatory T cells in pancreatic cancer express the αvβ5 integrin as a targetable activation marker

doi: 10.1101/2023.05.24.542137

Figure Lengend Snippet: a, b Naïve CD4 + T cells isolated from healthy mouse spleens were expanded in the presence of anti-CD3/CD28 Abs and TGF-β1 ( a ) or anti-CD3/CD28 Abs alone ( b ) for 3 days. The resulting populations were gated based on Foxp3 and CD25 expression (left panels). αvβ5 integrin and NRP-1 expression on Foxp3 + cells ( a , top row) and Foxp3 neg cells ( a , bottom row; b ) was analyzed by flow cytometry. The red and blue boxes gate CD25 + and CD25 neg cells, respectively. The bar diagrams summarize the proportion of αvβ5 integrin-positive cells in the indicated T cell population. n = 3. Statistical analysis, Welch’s t test; p = 0.0021 ( a , top), p = 0.0014 ( a , bottom), p < 0.0001 ( b ). c Flow cytometric analysis showing the proportion of αvβ5 integrin + cells among CD4 + CD25 + Foxp3 + iTregs induced by increasing concentrations of anti-CD3 Ab. n = 3. Statistical analysis, one-way ANOVA; p = 0.0088 (1 vs 3), p = 0.0088 (1 vs 10). d-f Naïve CD4 + T cells were stimulated with anti-CD3/CD28 Abs and TGF-β1 in the absence or presence of iRGD or iRGE. Flow cytometry was performed to quantify the proportion of CD4 + CD25 + Foxp3 + iTregs ( d ) and αvβ5 integrin + cells among the iTregs ( e ). Apoptosis of αvβ5 integrin + iTregs was quantified by measuring cleaved caspase 3 using flow cytometry ( f ). n = 3. Statistical analysis, one-way ANOVA ( d , e ) or Welch’s t test ( f ); p = 0.0042 ( d , None vs iRGD), p = 0.1774 ( d , None vs iRGE), p = 0.0027 ( e , None vs iRGD), p = 0.0529 ( e , None vs iRGE), p = 0.001 ( f ). Error bars, mean ± standard error; * p < 0.05; ** p < 0.01; **** p < 0.0001; N.S., not significant.

Article Snippet: After removing dead cells with a Dead Cell Removal kit (Miltenyi Biotec), the cells were treated with a PE-conjugated anti-CD25 Ab (Miltenyi Biotec), PE-conjugated anti-CCR8 Ab (eBioscience), and/or Alexa Fluor ® 647-conjugated anti-β5 integrin Ab (clone ALULA; BD Biosciences) combined with an anti-PE or -AF647 magnetic separation technique (Miltenyi Biotec) to enrich for CCR8 + iTregs, αvβ5 integrin + CCR8 + iTregs, or αvβ5 integrin neg CCR8 + iTregs.

Techniques: Isolation, Expressing, Flow Cytometry

Journal: bioRxiv

Article Title: Tumor-resident regulatory T cells in pancreatic cancer express the αvβ5 integrin as a targetable activation marker

doi: 10.1101/2023.05.24.542137

Figure Lengend Snippet: a CD4 + CD25 + Foxp3 + T cells (nTregs) were enriched from the spleens of healthy C57B6129SF1/J hybrid mice by magnetically removing CD4 neg T cells and CD25 neg T cells. The middle two panels show the expression of αvβ5 integrin and NRP-1 on the nTregs (red box) and naïve CD4 + CD25 neg Foxp3 neg T cells (blue box) analyzed by flow cytometry. The bar diagram summarizes the proportion of αvβ5 integrin + cells among the two populations. n = 3. Statistical analysis, Welch’s t test; p = 0.4721. b The pool in ( a ) was treated with anti-CD3/CD28 Abs alone for 3 days (left panel). αvβ5 integrin and NRP-1 expression on Foxp3 + cells (top row) and Foxp3 neg cells (bottom row) was analyzed by flow cytometry. The red and blue boxes gate CD25 + and CD25 neg cells, respectively. The bar diagrams summarize the proportion of αvβ5 integrin + cells among the indicated T cell populations. n = 3. Statistical analysis, Welch’s t test; p = 0.0064 (top), p = 0.0035 (bottom). Error bars, mean ± standard error; ** p < 0.01; N.S., not significant.

Article Snippet: After removing dead cells with a Dead Cell Removal kit (Miltenyi Biotec), the cells were treated with a PE-conjugated anti-CD25 Ab (Miltenyi Biotec), PE-conjugated anti-CCR8 Ab (eBioscience), and/or Alexa Fluor ® 647-conjugated anti-β5 integrin Ab (clone ALULA; BD Biosciences) combined with an anti-PE or -AF647 magnetic separation technique (Miltenyi Biotec) to enrich for CCR8 + iTregs, αvβ5 integrin + CCR8 + iTregs, or αvβ5 integrin neg CCR8 + iTregs.

Techniques: Expressing, Flow Cytometry

Journal: bioRxiv

Article Title: Tumor-resident regulatory T cells in pancreatic cancer express the αvβ5 integrin as a targetable activation marker

doi: 10.1101/2023.05.24.542137

Figure Lengend Snippet: Naïve CD4 + CD25 neg Foxp3 neg T cells were magnetically isolated from the spleens of healthy C57B6129SF1/J mice. The cells were stimulated with anti-CD3/CD28 Abs and TGF-β1 for 3 days to induce CD4 + CD25 + Foxp3 + iTregs. a-c Expression of CCR8 and αvβ5 integrin on the T cells before and after the stimulation. Representative dot plots from 3 or 4 separate studies are shown in ( a ). The bar diagram in ( b ) summarizes the proportion of CCR8 + cells (white bars) and αvβ5 integrin + cells (black bars) among each T cell population in ( a ). The Venn diagram in ( c ) summarizes the proportion of iTregs that expressed CCR8 and/or αvβ5 integrin. d-f Treg suppression assays were performed by co-culturing iTregs and Tconv (CD4 + and CD8 + ) at a 1 : 4 ratio in the presence of anti-CD3/CD28 Abs (TCR stimulation) for 3 days. We used iTregs that were enriched for CCR8 + iTregs ( d ) or CCR8 + iTregs that were either depleted or enriched for αvβ5 integrin + cells ( e ). The expression of CCR8 and αvβ5 integrin on the iTregs is shown in the representative dot plots. Proliferation of Tconv was analyzed by flow cytometry using Cell Trace Violet as shown in the representative histograms: Shaded, iTregs + Tconv (with TCR stimulation); black solid line, Tconv alone (with TCR stimulation); black dotted line, Tconv alone (no TCR stimulation). The bar diagrams in ( f ) summarize the values from ( d ) and ( e ) normalized to stimulated Tconv alone. n = 3. Statistical analysis, one-way ANOVA; p < 0.0001 ( b , CCR8 + , CD25 neg Foxp3 neg vs CD25 + Foxp3 neg and CD25 + Foxp3 neg vs CD25 + Foxp3 + ; αvβ5 + , CD25 + Foxp3 neg vs CD25 + Foxp3 + ), p = 0.0989 ( b , αvβ5 + , CD25 neg Foxp3 neg vs CD25 + Foxp3 neg ), p = 0.0003 ( f , CD4, CCR8 + vs αvβ5 neg CCR8 + ), p = 0.0002 ( f , CD4, CCR8 + vs αvβ5 + CCR8 + ), p < 0.0001 ( f , CD4, αvβ5 neg CCR8 + vs αvβ5 + CCR8 + ), p = 0.1048 ( f , CD8, CCR8 + vs αvβ5 neg CCR8 + ), p < 0.0001 ( f , CD8, CCR8 + vs αvβ5 + CCR8 + and αvβ5 neg CCR8 + vs αvβ5 + CCR8 + ). Error bars, mean ± standard error; *** p < 0.001; **** p < 0.0001; N.S., not significant.

Article Snippet: After removing dead cells with a Dead Cell Removal kit (Miltenyi Biotec), the cells were treated with a PE-conjugated anti-CD25 Ab (Miltenyi Biotec), PE-conjugated anti-CCR8 Ab (eBioscience), and/or Alexa Fluor ® 647-conjugated anti-β5 integrin Ab (clone ALULA; BD Biosciences) combined with an anti-PE or -AF647 magnetic separation technique (Miltenyi Biotec) to enrich for CCR8 + iTregs, αvβ5 integrin + CCR8 + iTregs, or αvβ5 integrin neg CCR8 + iTregs.

Techniques: Isolation, Expressing, Flow Cytometry

Journal: PLoS Pathogens

Article Title: Serum bridging molecules drive candidal invasion of human but not mouse endothelial cells

doi: 10.1371/journal.ppat.1010681

Figure Lengend Snippet: (A-F) Effects of inhibiting αv integrin function with siRNA knockdown (A and B or)specific monoclonal antibodies (C-F) and on the endocytosis (A, C, D) and cell-association (B, E, F) of serum-coated C . glabrata . (G and H) Inhibition of gC1qR (with monoclonal antibody 74.5.2) and αv integrins has an additive effect on decreasing the endocytosis (G) but not cell-association of serum-coated C . glabrata (H). Results are the mean ± SD of 3 experiments, each performed in triplicate. Orgs/HPF, organisms per high power field; ns, not significant; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 by ANOVA with the Dunnett’s test for multiple comparisons (A, B, G, H) or the Student’s t-test (C-F).

Article Snippet: For the cells transduced with hC1QBP, 10 μg/ml of blasticidin (Gibco; # A1113903) was added to the medium 2 d post transduction to select for transduced cells and selection was maintained for 7 d. Expression of eGFP was determined by fluorescent microscopy and expression of gC1qR, integrin αv, and integrin β5 were verified via immunoblotting of whole cell lysates with an anti-gC1qR antibody (clone 60.11), anti- integrin αv antibody (MilliporeSigma; #AB1930), and anti- integrin β5 antibody (My Biosource, Inc; # MBS617750).

Techniques: Inhibition

Journal: PLoS Pathogens

Article Title: Serum bridging molecules drive candidal invasion of human but not mouse endothelial cells

doi: 10.1371/journal.ppat.1010681

Figure Lengend Snippet: (A and B) Endocytosis of C . glabrata coated with either human or mouse serum by the indicated endothelial cells after 45 min (A) and 180 min (B). (C) Endocytosis of C . glabrata coated with fresh human serum by mouse liver endothelial cells expressing human gC1qR, integrin αv, or integrin β5. Data are the mean ± SD of 3 experiments each performed in triplicate. HUVEC, human umbilical vein endothelial cell; orgs/HPF, organisms per high power field; ns, not significant; ** P < 0.01, **** P < 0.0001. *** P < 0.001, **** P < 0.0001 by ANOVA with the Dunnett’s test for multiple comparisons.

Article Snippet: For the cells transduced with hC1QBP, 10 μg/ml of blasticidin (Gibco; # A1113903) was added to the medium 2 d post transduction to select for transduced cells and selection was maintained for 7 d. Expression of eGFP was determined by fluorescent microscopy and expression of gC1qR, integrin αv, and integrin β5 were verified via immunoblotting of whole cell lysates with an anti-gC1qR antibody (clone 60.11), anti- integrin αv antibody (MilliporeSigma; #AB1930), and anti- integrin β5 antibody (My Biosource, Inc; # MBS617750).

Techniques: Expressing

Journal: Science Advances

Article Title: Direct targeting of oncogenic RAS mutants with a tumor-specific cytosol-penetrating antibody inhibits RAS mutant–driven tumor growth

doi: 10.1126/sciadv.aay2174

Figure Lengend Snippet: ( A ) Engineering procedures to develop a second-generation active RAS-specific iMab, inRas37. ( B and C ) Elution profiles of antibodies purified on a size exclusion Superdex ( B ) and hydrophobic Zenix ( C ) column, with monitoring at 280 nm. The arrows indicate the elution positions of molecular weight standards. ( D ) Evaluation of nonspecific binding of the indicated antibodies to four antigens [double-stranded DNA (dsDNA), insulin, hemocyanin, and cardiolipin], as determined by ELISAs. ( E ) Selective binding of inRas37 (100 nM) to the GppNHp-bound active form of the indicated RAS proteins in comparison with the inactive GDP-bound form, as determined by an ELISA. ( F ) Binding specificity of the indicated antibodies (each at 20 nM) to integrin αvβ3 and integrin αvβ5 expressed on the surface of the indicated human cancer cells (flow cytometry data). siRNA, small interfering RNA. ( G ) Cellular internalization and cytosolic localization of the indicated antibodies (green) in the cells treated with 1 μM antibody for 12 hours before microscopic confocal analysis. Scale bars, 20 μm. ( H ) Split-GFP complementation assay to determine cytosolic localization of GFP11-SBP2–fused antibodies in SW480-SA-GFP1–GFP10 cells after 6-hour treatment with 1 μM antibody. Right: Quantified amount of cytosolic antibodies. Scale bar, 20 μm.

Article Snippet: An integrin β5 knockdown was implemented with two small interfering RNAs specific for integrin β5; they were synthesized by Bioneer Co. (Korea).

Techniques: Purification, Molecular Weight, Binding Assay, Comparison, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Small Interfering RNA