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: Cell death & disease

Article Title: PAK1 regulates RUFY3-mediated gastric cancer cell migration and invasion.

doi: 10.1038/cddis.2015.50

Figure Lengend Snippet: Figure 1 Overexpression of RUFY3 induces the formation of F-actin-enriched protrusion at the cell periphery. (a) GFP-RUFY3 localizes in F-actin-enriched invadopodia at the cell periphery of SGC-7901 cells. (Left panel) The living cell image acquisition was performed at 25 °C with SGC-7901 cells transfected with GFP-RUFY3 and undergoing a scratch wound assay, and GFP vector was used as a control. A representative image was shown. The white boxed areas in the left images (×100; scale bars, 200 μm) are magnified in the right images (×600; scale bars, 24 μm). The red boxed area in the right images shows that the cells expressing GFP-RUFY3 can localize at the periphery in a scratch area. (Right panel) Histogram showed the relative percentage of cells with actin protrusion at the migrating edge. Data are the average of at least three independent experiments with similar results, in which ~ 100 cells were counted (**Po0.01, compared with GFP vector). Protein expression was confirmed by western blotting assays using GFP-tagged antibody when equal glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the endogenous reference protein. (b and c) RUFY3 colocalizes with F-actin at the cell periphery. SGC-7901 cells were transiently transfected with pEGFP-C1 or pEGFP-RUFY3. Rhodamine-conjugated phalloidin was used to detect F-actin. After 24 h transfection, cells were fixed and permeabilized. (b) Images were captured using a scanning confocal fluorescence microscope and one confocal section is shown in each image. Scale bars, 10 μm. (c) Histogram showed the relative percentage of colocalization cells expressing GFP-RUFY3 with F-actin at the cell periphery. The data show mean ± S.E.M. (**Po0.01, compared with GFP vector), in which ~ 40 transfected cells were observed. (d) Colocalization of GFP-RIPX and myosinIIb at the cell periphery is shown by confocal microscopy. SGC-7901 cells were transiently transfected with GFP vector or GFP-RIPX. Colocalization of myosinIIb (red) with GFP-RIPX is shown by yellow fluorescence. Scale bars, 10 μm. (e) Colocalization of GFP-RIPX and integrin β5 at the cell periphery by plating cells on vitronectin is observed by confocal microscopy. SGC-7901 cells were transiently transfected with GFP vector or GFP-RIPX. Colocalization of integrin β5 (red) with GFP-RIPX is shown by yellow fluorescence. Scale bars, 20 μm. (f) Colocalization of GFP-RIPX and integrin α3β1 at the cell periphery by plating cells on vitronectin is observed by confocal microscopy. Scale bars, 20 μm

Article Snippet: The membrane was blocked with 5% nonfat dry milk in TBS-T (20 mM Tris, pH 7.4, 137 mM NaCl, 0.05% Tween-20) for 3 h at room temperature, and the proteins were probed with specific antibodies: GFP and His (GenScript Corporation, Nanjing, China), Flag (Shanghai Kangcheng), PAK1, integrin β5 and myosinIIb (Cell Signaling), RUFY3 and vinculin (Santa Cruz), integrin α3β1 (Bioss Inc. and Abcam).

Techniques: Over Expression, Transfection, Scratch Wound Assay Assay, Plasmid Preparation, Control, Expressing, Western Blot, Fluorescence, Microscopy, Confocal 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 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: bioRxiv

Article Title: Fibrin fragment E potentiates TGF-β-induced myofibroblast activation and recruitment

doi: 10.1101/829945

Figure Lengend Snippet: Interactions between soluble recombinant αVβ 3 (5-50 nM) and immobilized FnE (A) or FnDD (B) were determined by SPR. Immobilization was at 952 RU and 1251 RU respectively. (C) SPR sensorgrams of the interaction between FnE and integrin αVβ 3 fitted to a bivalent analyte model (i), a heterogeneous ligand model (ii) or to the 1:1 two state interaction model (iii). (D) Representative western blot and (E) quantification of α-SMA protein levels of HFL1 fibroblasts in the presence of the cyclic-RGD peptide EMD 66203 in combination with TGF-β (5 ng/ml) and/or FnE (2 nM). α-SMA was normalized against β-actin and expressed as compared to vehicle (DMSO) treated controls. (F) Chemotaxis and (G) net migration of HFL1 cells in presence of 5 µM cyclic-RGD peptide. Dashed grey lines indicate mean values for cells without addition of cyclic-RGD. (H) Integrin β3 and β5 protein levels in HFL1 cells after transfection with siRNA selectively targeting these integrins, as indicated. (I) α-SMA ( ACTA2 ) mRNA levels of siRNA transfected cells stimulated with TGF-β (5 ng/ml) with or without addition of FnE (2 nM) for 48 h. (J) Chemotaxis and (K) net migration of HFL1 cells treated with siRNA to suppress integrin β3 or β5 in response to a PDGF-BB (0-20 ng/ml) or FnE (0-2 nM) gradients. * p<0.05. Error bars represent standard error of the mean ( n ≥3).

Article Snippet: Primary antibodies used were mouse anti-α-SMA (clone 1A4, Sigma-Aldrich) 1:10 000, rabbit anti-β-actin (ab8227, Abcam) 1:5000, mouse anti-PARP1 (BD-bioscience) 1:1500, mouse anti-integrin β3 (Cell signaling) 1:500, rabbit anti-integrin β5 (Abnova) 1:250, all added in blocking buffer with 0.1% Tween-20.

Techniques: Recombinant, Western Blot, Chemotaxis Assay, Migration, Transfection

Journal: bioRxiv

Article Title: Identification of Serum Bridging Molecules that Mediate Human Endothelial Cell Invasion by Candida species

doi: 10.1101/2021.09.18.460925

Figure Lengend Snippet: Integrins αvβ3 and αvβ5 are endothelial cell receptors for serum-coated C. glabrata . (A-F) Effects of inhibiting αv integrin function with specific monoclonal antibodies (A-D) and siRNA knockdown (E-F) on the endocytosis (A, B, E) and cell-association (C, D, 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). (I) Confocal micrographs showing the accumulation of gC1qR and integrins αvβ3 and αvβ5 around serum-coated C. glabrata cells. Representative results of 3 independent experiments. Arrows indicate the C. glabrata cells and the endothelial cell receptors that have accumulated around them. Scale bar, 7 μm. Results shown in (A-H) 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.

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: bioRxiv

Article Title: Identification of Serum Bridging Molecules that Mediate Human Endothelial Cell Invasion by Candida species

doi: 10.1101/2021.09.18.460925

Figure Lengend Snippet: Mouse endothelial cells poorly support bridging molecule-mediated endocytosis. (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.

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