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: 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: bioRxiv

Article Title: Daam1 negatively regulates USP10 activity

doi: 10.1101/2022.02.14.480271

Figure Lengend Snippet: A) HCFs were transfected with siDaam1 or siGLO and treated with TGFβ for 3 days to increase integrin expression. siDaam1 transfection resulted in increased expression of αv-, β1-, β5-integrins. N=6. B) HCFs were transfected with siDaam1 or siGLO. After 3 days cells were lysed and subjected to Ubiquant™ ubiquitin capture ELISA. siDaam1 reduced, whereas siUSP10 increased ubiquitination of β1 and β5. N=4. (C) HCFs were transfected with siGLO or siDaam1 and treated with TGFβ for 3 days prior to processing for qPCR. The relative expression of Daam1, integrin αV, β1, and β5 is shown as compared to GAPDH. Statistical significance was calculated using an unpaired t-test Bar=200μm. N=3.

Article Snippet: Wells were then washed 4 times with TBST and then incubated in 1x Blocking Buffer in PBS with a 1:10,000 dilutions of either β1- (Assay Biotechnology; San Francisco, CA; catalogue #: R12-2927) or β5-integrin (Assay Biotechnology; San Franscisco, CA; catalogue #: F-5) primary antibody for 1 hr at room temperature.

Techniques: Transfection, Expressing, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Daam1 negatively regulates USP10 activity

doi: 10.1101/2022.02.14.480271

Figure Lengend Snippet: A) Live cell integrin recycling assay. 48 hrs post-transfection cells were blocked and treated with Ab against α5β1 or αv at 10ug/ml for 30 minutes prior to cell surface stripping for 30 sec. Cells were incubated for 90 min prior to incubation with 2° Ab-488 for 30 min. Bar=50μm. Quantification of recycled (B) Integrin α5β1 and (C) Integrin αV, N=3 with a total of 15 images analyzed for each condition. (D) Live cell FN recycling assay. HCFs were transfected as stated above. 24hrs post transfection HCFs were loaded with biotinylated-FN for 3 hours. After trypsinization (to separate cells from extracellular, non-internalized FN), HCFs were replated and imaged 48 hours post transfection. Prior to imaging, cells were incubated with streptavidin-488 to detect only recycled biotinylated-FN. Imaged by live cell confocal. Bar= 50μm. N=3. D) Quantification of extracellular FN. A total of 15 cells were analyzed for each condition from 3 independent experiments. E) Cell numbers 2 days after transfection for each condition are not significantly different. N=3. Image analysis was done using ImageJ’s Analyze Particles function. Statistical significance was calculated using an unpaired t-test +/- SEM.

Article Snippet: Wells were then washed 4 times with TBST and then incubated in 1x Blocking Buffer in PBS with a 1:10,000 dilutions of either β1- (Assay Biotechnology; San Francisco, CA; catalogue #: R12-2927) or β5-integrin (Assay Biotechnology; San Franscisco, CA; catalogue #: F-5) primary antibody for 1 hr at room temperature.

Techniques: Transfection, Stripping Membranes, Incubation, Imaging

Journal: bioRxiv

Article Title: Daam1 negatively regulates USP10 activity

doi: 10.1101/2022.02.14.480271

Figure Lengend Snippet: In response to stress-induced TGFβ1 release, both USP10 and Daam1 expression is increased. Daam1 sequesters USP10 to stress fibers, and USP10’s DUB activity on integrins is inhibited by Daam1. This may act as a level of control over USP10 activity in myofibroblasts. We further hypothesize that the known interaction of USP10 and G3BP1/2 proteins (also shown in ) positively regulates USP10’s DUB activity on integrins leading to integrin accumulation. The previously known roles of G3BP1/2 in integrin signaling support this concept.

Article Snippet: Wells were then washed 4 times with TBST and then incubated in 1x Blocking Buffer in PBS with a 1:10,000 dilutions of either β1- (Assay Biotechnology; San Francisco, CA; catalogue #: R12-2927) or β5-integrin (Assay Biotechnology; San Franscisco, CA; catalogue #: F-5) primary antibody for 1 hr at room temperature.

Techniques: Expressing, Activity Assay