Journal: Nature Communications

Article Title: Tumor-penetrating therapy for β5 integrin-rich pancreas cancer

doi: 10.1038/s41467-021-21858-1

Figure Lengend Snippet: a Dot plots representing uptake of Alexa 647-labeled iRGD-AgNPs or control AgNPs by mCherry-labeled CAF spheroids. The bar diagram shows the proportion of CAFs that internalized the AgNPs. n = 4 independent experiments. Two-tailed unpaired t test; p = 0.0008. b Dot plots representing iRGD-AgNP uptake by mCherry-labeled CAF spheroids in the presence of anti-NRP-1, αvβ3, or αvβ5 antibodies or control IgG. The bar diagram shows the proportion of CAFs that internalized the AgNPs normalized against IgG. n = 4 (Rabbit IgG and NRP-1), n = 5 (Mouse IgG, αvβ3, and αvβ5) independent experiments. One-way ANOVA; p = 0.4784 (NRP-1 vs. αvβ3), p < 0.0001 (NRP-1 vs. αvβ5 and αvβ3 vs. αvβ5). c Representative confocal images from three independent experiments of GFP-positive hPCF1424 CAFs (green) treated with Alexa 647-labeled iRGD-AgNPs (red) in the presence of mouse IgG (left panels) or an anti-αvβ5 blocking antibody (right panels). The cells were etched to remove the AgNPs bound to the surface and highlight the internalized particles. Pre-etch and post-etch images are shown. Scale bars 100 µm. d Bar diagram showing the % expression of αvβ5 or NRP-1 in hPCF1424 CAFs transiently transfected with non-specific siRNA (NS), two different pools of siRNAs against integrin β5 (ITGb5-1 and -2), or NRP-1 siRNA (siNRP-1), as measured by median fluorescence intensity (left panel). Representative data of three biological replicates. e Bar diagram showing flow cytometric analysis of iRGD-AgNP uptake by the siRNA-treated CAFs in ( d ). The results are shown as the proportion of CAFs that internalized the AgNPs. n = 3 (NRP-1), n = 5 (ITGb5-1 and -2) independent experiments. One-way ANOVA; p < 0.0001 (NS vs. siITGb5-1 and NS vs. siITGb5-2), p = 0.9997 (NS vs. siNRP-1). All error bars, SEM. *** p < 0.001; **** p < 0.0001. Source data provided in Source Data file.

Article Snippet: The sections were treated with a rabbit anti-αvβ5 polyclonal antibody (1:200) (Cat. no. bs-1356R, BIOSS, Woburn, MA) or a rabbit polyclonal anti-cleaved caspase 3 antibody (1:250) (Cat. no. 9579S, Cell Signaling) at 4 °C overnight and incubated with ImmPRESS (IHC-P, rabbit, MP-7401) secondary antibody reagent (Vector, Burlingame, CA).

Techniques: Labeling, Two Tailed Test, Blocking Assay, Expressing, Transfection, Fluorescence

Journal: Nature Communications

Article Title: Tumor-penetrating therapy for β5 integrin-rich pancreas cancer

doi: 10.1038/s41467-021-21858-1

Figure Lengend Snippet: a Flow cytometry analysis showing entry of iRGD-AgNP or control AgNP into spheroids made of PC3 tumor cells alone or PC3 cells mixed with mCherry-labeled hPCF1424 CAFs (co-cult). Note that iRGD-AgNPs entered PC3 cells more efficiently in the presence of CAFs. b Quantified results of ( a ). Proportion of cells that internalized the particles are shown. n = 5 independent experiments. Two-tailed unpaired t test; p = 0.00015 (PC3 alone vs co-cult), p = 0.0177 (CAF alone vs. co-cult). c Flow cytometry analysis showing the expression of αvβ5 and αvβ3 integrins and NRP-1 in PC3 and CAF spheroids cultured alone (gray bars) or co-cultured with each other (black bars). n = 6 independent experiments. Two-tailed unpaired Student’s t test; p = 0.000325 (αvβ5: PC3 alone vs. co-cult), p = 0.0247 (αvβ5: CAF alone vs. co-cult), p = 0.0158 (αvβ3: PC3 alone vs. co-cult), p = 0.0132 (αvβ3: CAF alone vs. co-cult), p = 0.947 (NRP-1: PC3 alone vs. co-cult), p = 0.055 (CAF alone vs. co-cult). All error bars, SEM. * p < 0.05; *** p < 0.001. Source data provided in Source Data file.

Article Snippet: The sections were treated with a rabbit anti-αvβ5 polyclonal antibody (1:200) (Cat. no. bs-1356R, BIOSS, Woburn, MA) or a rabbit polyclonal anti-cleaved caspase 3 antibody (1:250) (Cat. no. 9579S, Cell Signaling) at 4 °C overnight and incubated with ImmPRESS (IHC-P, rabbit, MP-7401) secondary antibody reagent (Vector, Burlingame, CA).

Techniques: Flow Cytometry, Labeling, Two Tailed Test, Expressing, Cell Culture

Journal: Nature Communications

Article Title: Tumor-penetrating therapy for β5 integrin-rich pancreas cancer

doi: 10.1038/s41467-021-21858-1

Figure Lengend Snippet: a Expression of αvβ5 integrin in PC3 cells after 2 days of incubation in normal media (NM) or CM prepared from cultured hPCF1424 CAFs ( n = 9), mPCFAA0779 CAFs ( n = 7), or MIA PaCa-2 human PDAC cells ( n = 4) performed in independent experiments. Fold over NM is shown. One-way ANOVA; p < 0.0001 (NM vs. hPCF1424 CM), p = 0.5085 (NM vs. mPCFAA0779 CM), p = 0.9886 (NM vs. MIA PaCa-2). b Dot plots representing iRGD-AgNP or control AgNP uptake in PC3 cells cultured in NM or CM from hPCF1424 CAFs. c The bar diagrams show the proportion of PC3 (left) or LM-PmC (right) cells that internalized iRGD-AgNPs. The cells were incubated in NM or CM from hPCF1424 CAFs for 2 days prior to study. n = 6 (PC3), n = 3 (LM-P) independent experiments. Two-tailed unpaired Student’s t test; p = 0.0001 (PC3 NM vs. CAF CM), p = 0.0127 (LM-P NM vs. CAF CM). d Expression of actin, αv, and β5 integrin mRNAs normalized against cyclophilin A analyzed by qPCR in PC3 cells incubated with NM or CM from hPCF1424 CAFs. n = 3 independent experiments. Two-tailed unpaired Student’s t test; p = 0.00016 (αv), p < 0.0001 (β5), p = 0.923 (actin). e , f Expression of αvβ5 integrin ( e ) or NRP-1 ( f ) in PC3 cells cultured in normal media (NM) or CM from hPCF1424 CAFs in the presence or absence of exogenous TGF-β, a TGF-β-specific inhibitor LY2157299, or vehicle alone. Mean fluorescence intensity (MFI) assessed by flow cytometry is shown. n = 4 independent experiments. Two-tailed unpaired Student’s t test; p = 0.0008 ( e: NM vs. TGF-β), p = 0.0009 ( e: TGF-β vs. TGF-β + LY2157299), p = 0.7082 ( e: CAF CM vs. CAF CM + DMSO), p = 0.0177 ( e: CAF CM + DMSO vs. CAF CM + LY2157299). No significant differences in ( f ). All error bars, SEM; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. Source data provided in Source Data file.

Article Snippet: The sections were treated with a rabbit anti-αvβ5 polyclonal antibody (1:200) (Cat. no. bs-1356R, BIOSS, Woburn, MA) or a rabbit polyclonal anti-cleaved caspase 3 antibody (1:250) (Cat. no. 9579S, Cell Signaling) at 4 °C overnight and incubated with ImmPRESS (IHC-P, rabbit, MP-7401) secondary antibody reagent (Vector, Burlingame, CA).

Techniques: Expressing, Incubation, Cell Culture, Two Tailed Test, Fluorescence, Flow Cytometry

Journal: The Journal of Cell Biology

Article Title: Myofibroblast contraction activates latent TGF-β1 from the extracellular matrix

doi: 10.1083/jcb.200704042

Figure Lengend Snippet: Activation of latent TGF-β1 by Mf contraction is integrin mediated. Cultured rat lung Mfs express β3 (A), β1 (B), and αvβ5 integrin (C), as shown by confocal micrographs produced from immunostaining. (D) TMLC were seeded directly on 7-d-old Mf cultures 2 h before adding modulators of integrin binding for an additional 2 h; this was followed by another 1-h incubation in the absence or presence of 0.5 U/ml thrombin. Integrin binding was stimulated with Mn 2+ and inhibited using RGD peptides, LAP–TGF-β1 (LAPβ1) decapeptide, and integrin function-blocking antibodies; scrambled DGR peptide was used as control. The broken line shows basal latent TGF-β1 activation by Mfs; the dotted line demonstrates active TGF-β1 levels after inducing contraction with thrombin only. Results are expressed as a percentage of nonstimulated control (BSA) corrected for TMLC baseline reporter activity. (E) Controls were performed to test the effect of integrin modulators on TMLC reporter activity with and without stimulation by TGF-β1 and 0.5 U/ml thrombin. Error bars represent the SD of the mean. Bar, 10 μm.

Article Snippet: Function-blocking antibodies against β1 integrin (rabbit, AB1937; Millipore), β3 integrin (mouse clone F11; BD Biosciences), and αvβ5 integrin (mouse clone P1F6; Abcam) were added at 10 μg/ml for 3 h. Integrin binding to LAP was inhibited with 100 μg/ml of a custom-synthesized decapeptide containing the RGD sequence and flanking amino acids of rat LAP (KRRGDLSTIH).

Techniques: Activation Assay, Cell Culture, Produced, Immunostaining, Binding Assay, Incubation, Blocking Assay, Control, Activity Assay

Journal: The Journal of Cell Biology

Article Title: Myofibroblast contraction activates latent TGF-β1 from the extracellular matrix

doi: 10.1083/jcb.200704042

Figure Lengend Snippet: Integrin-transmitted contraction of TX-100 cytoskeletons activates latent TGF-β1. Cytoskeletons from 7-d-old Mf cultures were produced by cell extraction with TX-100. (A) Extraction efficiency was monitored by Western blotting for the plasma membrane–associated Na + /K + -ATPase. Stress fibers remaining after TX-100 extraction (B) were contracted by adding ATP for 30 min (C). Kymograph lines, 150 μm. Bar, 25 μm. (D) Isolated stress fiber contraction was visualized by a kymograph produced along the overlaid white lines in A and B. (E) TX-100 cytoskeletons were incubated for 2 h with protease inhibitors and integrin antagonists and subsequently contracted with ATP for 1 h; supernatants were analyzed for their content in active TGF-β1. Results are expressed as the percentage of nonstimulated control (BSA) corrected for TMLC baseline reporter activity. (F) To control for the influence of ATP on TMLC reporter activity, TMLC were incubated with ATP for 1 h in the presence (dashed bars) or absence (solid bars) of TGF-β1. Error bars represent the SD of the mean.

Article Snippet: Function-blocking antibodies against β1 integrin (rabbit, AB1937; Millipore), β3 integrin (mouse clone F11; BD Biosciences), and αvβ5 integrin (mouse clone P1F6; Abcam) were added at 10 μg/ml for 3 h. Integrin binding to LAP was inhibited with 100 μg/ml of a custom-synthesized decapeptide containing the RGD sequence and flanking amino acids of rat LAP (KRRGDLSTIH).

Techniques: Produced, Extraction, Western Blot, Clinical Proteomics, Membrane, Isolation, Incubation, Control, Activity Assay

Journal: Journal of extracellular vesicles

Article Title: MFGE-8, a Corona Protein on Extracellular Vesicles, Mediates Self-Renewal and Survival of Human Pluripotent Stem Cells.

doi: 10.1002/jev2.70056

Figure Lengend Snippet: FIGURE 5 MFGE-8 facilitates EV uptake into recipient hESCs through integrin αvβ5. (a,b) The uptake of PKH-labelled EVs (30 µg/mL) that were pre-incubated with different concentrations of rhMFGE-8 (0–50 µg/mL) (a, n = 3) or anti-M8nAb (b, n = 4) for 1 h and then exposed to hESCs. The mean fluorescence intensity of hESC colonies was measured after 6 h. Cells were stained for OCT4 and E-cadherin, and nuclei were stained with DAPI. (c) Changes in the uptake of PKH-EVs (30 µg/mL) that were pre-incubated with or without rhMFGE-8 (25 µg/mL) for 1 h and exposed to hESCs for 6 h. anti- M8nAb (5 µg/mL) was added to the hESC culture for 1 h before the cells were exposed to PKH-EVs (n = 4). Cells were stained for OCT4 and E-cadherin, and nuclei were stained with DAPI. d, hESCs were exposed to untreated PKH-labelled bare EVs (30 µg/mL) or pre-treated for 3 h with hESC-derived SFs (bare EVs + SFs) or with hESC-derived SFs in which MFGE-8 activity was inhibited by anti-M8nAb (50 µg/mL). The mean fluorescence intensity was measured after 6 h of exposure (n = 4). Cells were stained for OCT4, and nuclei were stained with DAPI. (e) hESCs were exposed to PKH-EVs (30

Article Snippet: The following primary antibodies were used: mouse anti-OCT4 (1:400; sc-5279, Santa Cruz Biotechnology), rabbit anti-vimentin (1:200; 5741, Cell Signalling Technology), goat anti-E-cadherin (1:400; AF648, R&D Systems), mouse anti-integrin αvβ5 (1:100; MAB2528, R&D Systems), mouse anti-8-oxoguanine (1:200; MAB3560, Millipore) and rabbit anti-53BP1 (1:200; 4937, Cell Signalling Technology).

Techniques: Incubation, Fluorescence, Staining, Derivative Assay, Activity Assay

Journal: Journal of extracellular vesicles

Article Title: MFGE-8, a Corona Protein on Extracellular Vesicles, Mediates Self-Renewal and Survival of Human Pluripotent Stem Cells.

doi: 10.1002/jev2.70056

Figure Lengend Snippet: FIGURE 6 MFGE-8 mediates EV uptake in hESCs through the integrin αvβ5/Akt/GSK3β pathway. (a) A human phospho-kinase antibody array for hESCs cultured for 2 h in the absence or presence of anti-M8nAb (5 µg/mL) to block EV uptake. (b,c) Immunoblot showing phosphorylation of AKT and GSK3β after hESCs were treated with rhMFGE-8 (b) or hESC-derived EVs (c), with relative ratios of phosphorylation of AKT and GSK3β after 1 h of treatment shown on the right (n = 3). (d–f) Immunoblots showing the phosphorylation of AKT and GSK3β after hESCs were treated with different combinations of rhMFGE-8 (5 µg/mL), EVs (50 µg/mL), anti-M8nAb (5 µg/mL), anti-integrin αvβ5 antibody (anti-ITG αvβ5, 10 µg/mL), LY294002 (10 µM) and/or CHIR99021 (2 µM). hESCs were pre-incubated with or without anti-M8nAb, anti-integrin αvβ5 Ab, LY294002 and CHIR99021 for 1 h. Cells were treated with rhMFGE-8 or hESC-EVs for 15 min and 30 min, respectively. Relative phosphorylation is shown as a bar graph on the right (n = 3). (g) Image of PKH-EV uptake in hESCs treated with the factors indicated. PKH-labelled EVs (30 µg/mL) were pre-incubated with or without anti-M8nAb (25 µg/mL) for 1 h and exposed to hESCs for 6 h. The anti-ITG αvβ5 (10 µg/mL) or LY294002 (10 µM) was added to the hESC culture for 1 h before the cells were exposed to the PKH-EVs. Cells were stained for OCT4 and E-cadherin, and nuclei were stained with DAPI. Relative EV uptake is shown as a

Article Snippet: The following primary antibodies were used: mouse anti-OCT4 (1:400; sc-5279, Santa Cruz Biotechnology), rabbit anti-vimentin (1:200; 5741, Cell Signalling Technology), goat anti-E-cadherin (1:400; AF648, R&D Systems), mouse anti-integrin αvβ5 (1:100; MAB2528, R&D Systems), mouse anti-8-oxoguanine (1:200; MAB3560, Millipore) and rabbit anti-53BP1 (1:200; 4937, Cell Signalling Technology).

Techniques: Ab Array, Cell Culture, Blocking Assay, Western Blot, Phospho-proteomics, Derivative Assay, Incubation, Staining

Journal: Journal of extracellular vesicles

Article Title: MFGE-8, a Corona Protein on Extracellular Vesicles, Mediates Self-Renewal and Survival of Human Pluripotent Stem Cells.

doi: 10.1002/jev2.70056

Figure Lengend Snippet: FIGURE 7 MFGE-8 promotes endocytosis of EVs and self-renewal of hESCs by activating dynamin-1 and cyclin D1 via the integrin αvβ5/Akt/GSK3β axis. (a) Relative EV uptake in hESC cells treated with endocytosis inhibitors for 1 h (n = 3). hESC cells were incubated with PKH-EVs (30 µg/mL) for 6 h. (b) Images and quantitation of PKH-EV uptake in hESCs. EVs were pre-incubated with or without rhMFGE-8 (25 µg/mL) for 1 h before they were used to treat hESCs in the presence or absence of dynasore (DNS, 10 µM) (n = 4). Cells were stained for OCT4 and E-cadherin, and nuclei were stained with DAPI. (c) RT-qPCR analysis of DNM1 and DNM2 expression in hESCs (n = 3). (d) EV uptake in hESCs after siRNA knockdown of dynamin 1 (siDNM1) and dynamin 2 (siDNM2). Cell nuclei were stained with DAPI. Two siRNAs (#1 and #2) targeting different mRNA sequences of dynamins were used (n = 4). (e) Immunoblots showing phosphorylation of DNM1 in hESCs treated with or without 100 µg/mL hESC-EVs for 1 h. The relative phosphorylation of DNM1, quantitated by densitometry, is shown as a bar graph on the left (n = 3). (f) Images and quantitation of EV uptake for hESCs treated with combinations of anti-M8nAb and inhibitors, as indicated. hESCs were pre-treated with LY294002 (10 µM), CHIR99021 (2 µM), DNS (10 µM) or chlorpromazine (CPZ) (1 µM). hESC-EVs were pre-incubated with or without anti-M8nAb (25 µg/mL) for 1 h before exposure to cells (n = 3). Cells

Article Snippet: The following primary antibodies were used: mouse anti-OCT4 (1:400; sc-5279, Santa Cruz Biotechnology), rabbit anti-vimentin (1:200; 5741, Cell Signalling Technology), goat anti-E-cadherin (1:400; AF648, R&D Systems), mouse anti-integrin αvβ5 (1:100; MAB2528, R&D Systems), mouse anti-8-oxoguanine (1:200; MAB3560, Millipore) and rabbit anti-53BP1 (1:200; 4937, Cell Signalling Technology).

Techniques: Incubation, Quantitation Assay, Staining, Quantitative RT-PCR, Expressing, Knockdown, Western Blot, Phospho-proteomics