Journal: Cancers

Article Title: Are Integrins Still Practicable Targets for Anti-Cancer Therapy?

doi: 10.3390/cancers11070978

Figure Lengend Snippet: Selected clinical trials of agents targeting integrins. Non-exhaustive listing of the recent most important clinical studies with integrin inhibitors and their salient features and results).

Article Snippet: Cilengitide (EMD 121974, anti-αVβ3/αVβ5 integrin cyclic peptide) (Merck-Serono): total no. of trials: 21 (+ 8 terminated) , , , , , , .

Techniques: Activity Assay

Journal: The Journal of Cell Biology

Article Title: Giant worm-shaped ESCRT scaffolds surround actin-independent integrin clusters

doi: 10.1083/jcb.202205130

Figure Lengend Snippet: IST1-immunoprecipitation and proteomics. (A) moDCs were cultured either in the presence or absence of serum followed by IST1 immunoprecipitation. Proteins were identified by mass spectrometry. Data is obtained using four donors. A.I: Flowchart of the IST1 IP. A.II: Western blot of eluted IP sample probed for IST1. A.III: Venn diagram showing proteins consistently enriched ≥1.3-fold in comparison with the sample with beads only (no antibody). The blue circle represents proteins upregulated in the samples cultured with serum and the yellow circle represents proteins upregulated in the samples cultured without serum. A.IV: Protein network showing proteins ≥1.3 times enriched in the serum-free condition over the serum-containing condition. Using the STRING tool, set at a confidence level of 0.9, proteins associated with cell adhesion were selected. Size of the dots corresponds with enrichment of the protein in the absence of serum. (B) Western blot of the pulldown confirming immunoprecipitation of the target IST1. Interaction with integrin β2 could not be detected by Western blot (expected band missing). We could not exclude that the IST1 antibody binds to IgG heavy chain, as IST1 runs at nearly the same height as the heavy chain antibody (and protein A/G). However, we confirmed the presence of IST1 with mass spec in A using the exact same procedures. (C) Confocal micrographs of moDCs immunolabeled for integrin αVβ5 (cyan in merger), IST1 (magenta) and phalloidin (yellow). For this integrin, we only observed colocalization with the ESCRT structures in one out of four tested donors. Note that integrin αVβ5 also locates to podosomes (arrow). (D) TIRF microscopy of moDCs co-expressing mCherry-labeled CHMP4 (magenta) with YFP-tagged integrin β2 (green) or only YFP (control). Graphs show the average size number of CHMP4-positive structures for cells with detectable expression of both constructs for three different donors. NS: not significant (paired two-sided t test; data distribution was assumed to be normal, but this was not formally tested). Scale bars: 10 μm. Source data are available for this figure: .

Article Snippet: Integrin αVβ5 , Mouse , 1:200 , Merck Millipore , Clone 15F11 , .

Techniques: Immunoprecipitation, Cell Culture, Mass Spectrometry, Western Blot, Comparison, Immunolabeling, Microscopy, Expressing, Labeling, Control, Construct

Journal: The Journal of Cell Biology

Article Title: Giant worm-shaped ESCRT scaffolds surround actin-independent integrin clusters

doi: 10.1083/jcb.202205130

Figure Lengend Snippet: ESCRT structures surround actin-independent integrin clusters. (A) Confocal micrographs of monocyte-derived dendritic cells (moDCs) incubated for 40 min and immunolabeled for IST1 (magenta in merge), integrin αM or integrin β2 (cyan), and phalloidin (yellow). Blue: DAPI. Bar graphs show cells with integrin-wrapping IST1 structures as a percentage of all cells forming IST1 structures, at the indicated time points, for integrin β2 and integrin αM. Line graphs show fluorescence intensity profiles as indicated by the white line. ( n ≥ 3 donors; two-sided paired t tests; *: P < 0.05; **: P < 0.01; NS: not significant). Scale bars: 10 µm. (B) Immunofluorescence labeling of a temporal artery biopsy (same donor 1 as in ) for DAPI (blue), IST1 (magenta), integrin β2 (yellow), and macrophage marker CD68 or fibroblast marker vimentin (cyan). (C) Confocal micrographs of moDCs seeded in the presence or absence of 5 mM EDTA, and immunostained for IST1 (magenta), integrin β2 (green), and phalloidin (yellow). Arrows: IST1-positive structures. Graph: average number of IST1-positive structured per cell ( n = 3 donors). For the statistical analysis of A and C, data distribution was assumed to be normal, but this was not formally tested. Scale bars: 10 µm.

Article Snippet: Integrin αVβ5 , Mouse , 1:200 , Merck Millipore , Clone 15F11 , .

Techniques: Derivative Assay, Incubation, Immunolabeling, Fluorescence, Immunofluorescence, Labeling, Marker

Journal: The Journal of Cell Biology

Article Title: Giant worm-shaped ESCRT scaffolds surround actin-independent integrin clusters

doi: 10.1083/jcb.202205130

Figure Lengend Snippet: Latruculin B (LatB) control experiments and knockdowns of ESCRT proteins. (A) moDC preincubated in the presence of 100 μM LatB and cultured for 1 h in the absence of LatB. The cell is immunolabeled for IST1 (magenta), phalloidin (green), and DAPI (blue). No F-actin staining was observed in the presence of LatB, and phalloidin showed weak nuclear staining. Scale bar: 10 μm. (B) Left: Scheme of workflow of adhesion isolation. Right: Volcano plot of mass spectrometry hits. Key consensus adhesion proteins were depleted from adhesions in response to LatB treatment. (C.I) Western blot of moDCs treated with non-targeting (NT) siRNA, IST1 siRNA, or TSG101 siRNA. Total protein levels were measured using Ponceau S and the blot was probed for TSG101 and IST1. (C.II) Quantification of C.I. Protein levels were corrected for loading using Ponceau S intensities and subsequently normalized to the NT condition. Graphs show mean ± SD ( n ≥ 5 donors). (D) Effect of IST1 knockdown and TSG101 knockdown on the average number of IST1-positive structures per cell, the average size of the structures and the average total area covered by the structures per cell. Effects are shown both in untreated cells and in LatB treated cells. Each dot-line-dot pair represents 1 donor. *: P < 0.05; NS: not significant (paired two-sided t test). (E) Fold change in the number of adherent cells upon LatB treatment in moDCs with IST1 or TSG101 knockdown compared to cells treated with NT siRNA. (paired two-sided t test). (F) Quantification of integrin β2 signal in the ESCRT structures, based on immunofluorescence labeling of moDCs for integrin β2 and IST1. Knockdown of IST1 and TSG101 significantly reduced the level of integrin β2 ( n = 3 donors; paired two-sided t test; ***: P < 0.001). (G.I) Western blot and quantification of knockdown of TSG101 and ALIX. Note unsuccessful knockdown of ALIX. (G.II) Flow cytometry of surface levels of integrin αM and integrin β2 in moDCs. TSG101 knockdown significantly lowered surface levels of integrin β2 ( n = 3 donors; paired two-sided t test). A representative histogram is shown. (H) DAPI influx in moDCs was not affected by knockdown of TSG101. Scale bars, 10 μm. For all statistical analysis, data distribution was assumed to be normal, but this was not formally tested. Source data are available for this figure: .

Article Snippet: Integrin αVβ5 , Mouse , 1:200 , Merck Millipore , Clone 15F11 , .

Techniques: Control, Cell Culture, Immunolabeling, Staining, Isolation, Mass Spectrometry, Western Blot, Knockdown, Immunofluorescence, Labeling, Flow Cytometry

Journal: The Journal of Cell Biology

Article Title: Giant worm-shaped ESCRT scaffolds surround actin-independent integrin clusters

doi: 10.1083/jcb.202205130

Figure Lengend Snippet: IST1 structures are recruited to sites of membrane damage. (A) STRING protein network of consensus adhesion proteins identified by DAVID gene ontology that were depleted in response to 100 μM latrunculin B (Lat B) treatment. (B) Confocal micrographs of moDCs pre-incubated with or without Lat B. After washing away the Lat B containing medium, cells were seeded and incubated for 1 h followed by gentle washing with PBS to remove non-adherent cells. Left graph: average number of adhering moDCs. Right graph: average number of IST1 structures per cell. ( n = 5 donors; >80 cells per donors; paired 2-sided t test). (C) Confocal micrographs of moDCs cultured with and without serum and in the presence of 0.5 μg/ml DAPI. Graph shows fluorescent intensities of DAPI. Data points show individual donors (>50 cells per condition; paired two-sided t test; *: P < 0.05; **: P < 0.01; ****: P < 0.0001; NS: not significant). (D) Confocal micrographs showing recruitment of ESCRT structures to plasma membrane contact sites (arrow heads) with membrane-disrupting silica crystals. (E) Model scheme with proposed mechanism. Integrins and other known extracellular cargo proteins (CD63, GPI-anchored proteins, ubiquitinated proteins) are enriched in membrane domains surrounded by ESCRT structures. The cortical F-actin cytoskeleton is disassembled at these clusters. The integrin clusters tightly adhere to the extracellular substrate, making the surrounding membrane vulnerable to damage. This results in formation of the ESCRT structures. ESCRT repairs the membrane by shedding of damaged plasma membrane regions. For the statistical analysis of B and C, data distribution was assumed to be normal, but this was not formally tested. Scale bars: 10 μm.

Article Snippet: Integrin αVβ5 , Mouse , 1:200 , Merck Millipore , Clone 15F11 , .

Techniques: Membrane, Incubation, Gentle, Cell Culture, Clinical Proteomics

Journal: The Journal of Cell Biology

Article Title: Giant worm-shaped ESCRT scaffolds surround actin-independent integrin clusters

doi: 10.1083/jcb.202205130

Figure Lengend Snippet: Primary antibodies used for samples for confocal imaging

Article Snippet: Integrin αVβ5 , Mouse , 1:200 , Merck Millipore , Clone 15F11 , .

Techniques: Microscopy, Ubiquitin Proteomics, Labeling

Journal: Pulmonary Circulation

Article Title: Dichotomous role of integrin‐β5 in lung endothelial cells

doi: 10.1002/pul2.12156

Figure Lengend Snippet: Increased expression of integrin β 5 in lung tissue from PAH patients and rats with cHx + EC clone PH. (a) Representative immunohistochemistry shows enhanced integrin‐β5 staining in pulmonary arteries from PAH patients, including elevated expression in PAECs (arrows), compared to the faint integrin‐β5 staining in pulmonary arteries from control subjects. Scale bar: 50 μm. (b) PAECs from PAH patients show increased ITGB5 mRNA expression compared to control PAECs. n = 4 (control) and n = 5 (PAH). (c) Representative integrin‐β5 immunohistochemistry images show increased endothelial integrin‐β5 staining in cHx + CD117 + EC clone rats compared to faint endothelial staining in normoxia + CD117 + EC clone rats. Scale bars: 25 μm. (d) cHx + CD117 + EC clone rats had increased lung tissue mRNA level of Itgb5 . (e) Electric cell impedance sensing (ECIS) measurement of transendothelial electrical resistance indicates accelerated EC barrier formation in EC clones compared to control ECs. (f) Gene array analysis shows integrin mRNA expression switch with reduced expression of Itgb1 and Itgb8 , and upregulation of Itgb5, Itgb3 , and Itgav in CD117 + EC clones compared to control ECs. Note that the most consistent upregulation in all four clonal EC lines was for Itgb5 . PAH, pulmonary arterial hypertension; PH, pulmonary hypertension. * p < 0.05, *** p < 0.001.

Article Snippet: Slides for anti‐integrin‐ανβ5 antibody treated rats were sectioned, stained and scanned by Histowiz.

Techniques: Expressing, Immunohistochemistry, Staining, Control, Clone Assay

Journal: Pulmonary Circulation

Article Title: Dichotomous role of integrin‐β5 in lung endothelial cells

doi: 10.1002/pul2.12156

Figure Lengend Snippet: Inhibition of integrin α ν β 5 affects TGF‐β1‐induced endothelial‐to‐mesenchymal transition gene expression in rat lung CD117 + EC clones versus control ECs. (a–g) Comparison of mRNA expression in rat lung control ECs and CD117 + EC clones following treatment with a neutralizing anti‐integrin ανβ5 antibody (10 μg/ml) and/or TGF‐β1 (10 ng/ml). Abbreviations: Vehicle for TGF‐β1 (veh), transforming growth‐factor‐β1 (TGF‐β1), isotype antibody (iso) and neutralizing anti‐integrin‐ανβ5 antibody (itgανβ5). (a) increased baseline levels of Itgb5 in EC clones, but significant induction of Itgb5 in control ECs by TGF‐β1, which was ameliorated by antibody inhibition. (b) induction of Vwf by anti‐integrin ανβ5 antibody in control ECs. (c) Cnn1 was induced by TGF‐β1 in control ECs, with a trend towards reduction by anti‐integrin ανβ5 antibody. In EC clones, Cnn1 was induced by TGF‐β1. (d) Tagln was induced by TGF‐β1 in control ECs and EC clones, and anti‐integrin ανβ5 antibody treatment reduced this increased Tagln expression in control ECs (trend) and EC clones. (e) Twist1 was induced in control ECs by TGF‐β1, and EC clones had higher baseline expression of Twist1 . (f) Snai1 was induced by TGF‐β1 in control ECs, and anti‐integrin ανβ5 antibody decreased Snai1 expression in control ECs and EC clones. (g) Serpine1 was induced by TGF‐β1 in control ECs and EC clones, and anti‐integrin ανβ5 antibody reduced Serpine1 expression in control ECs. n = 3 per group. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: Slides for anti‐integrin‐ανβ5 antibody treated rats were sectioned, stained and scanned by Histowiz.

Techniques: Inhibition, Gene Expression, Clone Assay, Control, Comparison, Expressing

Journal: Pulmonary Circulation

Article Title: Dichotomous role of integrin‐β5 in lung endothelial cells

doi: 10.1002/pul2.12156

Figure Lengend Snippet: Inhibition of integrin‐ανβ5 mainly affects TGF‐β1‐induced endothelial‐to‐mesenchymal transition on the gene expression level in human lung microvascular ECs. (a–e) Comparison of mRNA expression in human lung microvascular ECs following treatment with a neutralizing anti‐integrin ανβ5 antibody (10 μg/ml) and/or TGF‐β1 (10 ng/ml) for 72 h. Abbreviations: Vehicle (veh), isotype antibody (iso), transforming growth factor‐β1 (ΤGF‐β1) and neutralizing anti‐integrin‐ανβ5 antibody (itgανβ5). (a) Induction of ITGB5 expression in TGF‐β1 + anti‐integrin ανβ5 antibody treated ECs. (b, c) TGF‐β1 treatment reduced VCAM1 and PECAM1 mRNA expression and was not affected by anti‐integrin ανβ5 antibody. (d) TGF‐β1 induced ACTA2 expression, and anti‐integrin ανβ5 antibody reduced TGF‐β1‐induced ΑCTA2 expression. (e) While TGF‐β1 induced a trend towards elevated SNAI1 expression, the addition of anti‐integrin ανβ5 antibody elevated SNAI1 expression significantly. (f) Representative immunoblots for VCAM1 (short isoform), α‐SMA, SM22α, SNAIL1, and β‐actin (loading control) for HLMVECs following treatment with a neutralizing anti‐integrin ανβ5 antibody (10 μg/ml) and/or TGF‐β1 (10 ng/ml) for 72 h. (g–j) VCAM1 (short isoform, G), α‐SMA (h), SM22α (i) and SNAIL1 (j) protein expression. n = 3 per group. In (h) p Values are shown to demonstrate a trend. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: Slides for anti‐integrin‐ανβ5 antibody treated rats were sectioned, stained and scanned by Histowiz.

Techniques: Inhibition, Gene Expression, Comparison, Expressing, Western Blot, Control

Journal: Pulmonary Circulation

Article Title: Dichotomous role of integrin‐β5 in lung endothelial cells

doi: 10.1002/pul2.12156

Figure Lengend Snippet: Inhibition of integrin ανβ5 reduces gap closure in human lung microvascular ECs. (a, b) Representative differential interference contrast (DIC) images (a) show reduced gap closure following 24 h treatment with anti‐integrin ανβ5 antibody and/or TGF‐β1. Scale bar: 200 μm. (b) Quantification of percent gap closure over time. n = 3 per group. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: Slides for anti‐integrin‐ανβ5 antibody treated rats were sectioned, stained and scanned by Histowiz.

Techniques: Inhibition

Journal: Pulmonary Circulation

Article Title: Dichotomous role of integrin‐β5 in lung endothelial cells

doi: 10.1002/pul2.12156

Figure Lengend Snippet: Inhibition of integrin‐ανβ5 promotes apoptosis in human lung microvascular ECs and rat lung CD117 + EC clones, but not in control rat lung ECs. (a, b) Representative dot plots of Annexin V (AV) and 7‐aminoactinomycin (7‐AAD) flow cytometry (a) and quantification of AV + 7‐AAD − (early apoptotic) human lung microvascular ECs (HLMVECs) following 24 h treatment with isotype antibody (10 μg/ml) or anti‐integrin‐ανβ5 (ITGανβ5) antibody (5 or 10 μg/ml). (c, d) Representative dot plots of AV and 7‐AAD flow cytometry (c) and quantification of AV + 7‐AAD − rat lung control ECs and CD117 + EC clones following 24 h treatment with isotype antibody or anti‐integrin‐aνβ5 antibody (10 μg/ml) and recombinant human TGF‐β1 (5 ng/ml) or vehicle. n = 3 per group. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Slides for anti‐integrin‐ανβ5 antibody treated rats were sectioned, stained and scanned by Histowiz.

Techniques: Inhibition, Clone Assay, Control, Flow Cytometry, Recombinant

Journal: Pulmonary Circulation

Article Title: Dichotomous role of integrin‐β5 in lung endothelial cells

doi: 10.1002/pul2.12156

Figure Lengend Snippet: Inhibition of integrin‐ανβ5 exaggerates pulmonary hypertension in the rat cHx + CD117 + EC clone model. Physiology and tissue harvest was performed on day 21. (a) Representative histology images show enhanced muscularization and occlusion in cHx + CD117 + EC clone rats treated with neutralizing anti‐integrin‐ανβ5 antibody (ανβ5) versus isotype control antibody (con) (100 μg/kg three times a week from day 1–21). Scale bar: 25 μm. (b) Right ventricular systolic pressure (RVSP). (c) Fulton index of right ventricular hypertrophy: (right ventricle weight (RV) versus left ventricle + septum weight [LV + S]). (d) Media wall thickness (MWT). (e) Fraction of fully and partially occluded pulmonary arteries and not occluded pulmonary arteries. (f) ratio of pulmonary artery acceleration time (PAAT) versus pulmonary ejection time (PET) as measured by echocardiography. (g) Tricuspid annular plane systolic excursion (TAPSE) as measured by echocardiography. (h) Cardiac output estimated by echocardiography. n = 4 per group. Data are shown as single values and mean ± SEM. n.s, not significant. * p < 0.05, *** p < 0.001.

Article Snippet: Slides for anti‐integrin‐ανβ5 antibody treated rats were sectioned, stained and scanned by Histowiz.

Techniques: Inhibition, Control

Journal:

Article Title: Characterization of Entry and Infection of Monocytic THP-1 cells by Kaposi's Sarcoma Associated Herpesvirus (KSHV): Role of Heparan Sulfate, DC-SIGN, Integrins and Signaling

doi: 10.1016/j.virol.2010.07.012

Figure Lengend Snippet: A. KSHV internalization in the presence of heparin and mannan. 1×106 THP-1 cells mock treated or pre-incubated with the indicated concentrations of mannan at 4°C were infected with 10 KSHV DNA copies per cell or heparin pretreated KSHV (100ug/ml). At different time points, uninternalized virus was removed by washing and trypsinization. Infected and mock infected cells were washed and internalized viral copy numbers were determined by real-time DNA PCR as described in the figure 1C/D legend. The data are represented as the percent inhibition of KSHV DNA internalized in comparison with cells incubated with virus alone. Each reaction was done in duplicate and each bar represents the mean ± SD of the results of three experiments. B. KSHV internalization in THP1 cells upon blocking DC-SIGN. THP1 cells incubated with mouse IgG or mouse anti-DC-SIGN mAb at 4° C for 1 h. The washed cells were incubated with KSHV (10 genome copies/cell) at 4° C for 1 h followed by at 37° C for 1 h. Uninternalized virus was removed by washing and trypsinization, internalized viral copy numbers were determined by real-time DNA PCR as described in the figure 1C/D legend. The data are represented as the percent inhibition of KSHV DNA internalization in comparison with IgG control. C. KSHV gene expression in the presence of soluble integrins. 1×106 THP-1 cells were infected with 30 KSHV DNA copies per cell of untreated virus or with virus pretreated with 10μg/ml of soluble α3β1, αvβ3, αvβ5, α5β1 and α4β7 integrins for 1 h at 37° C. At 2 h p.i. uninternalized virus was removed by washing and trypsinization and the infected cells were further incubated. At 24 h p.i., the cells were washed, RNA was isolated, ORF73 gene expression was examined as described under the figure 2B legend. ORF73 gene expression was represented as percent inhibition compared to control untreated KSHV infection.

Article Snippet: Rabbit anti-integrin α3 and β1 and soluble human integrins α3β1, αvβ3, αvβ5 and α5β1 were from Upstate Biotechnology, Lake Placid, N.Y. Recombinant human integrin α4β7 was from R&D systems, Minneapolis, MN.

Techniques: Incubation, Infection, Inhibition, Blocking Assay, Expressing, Isolation

Journal:

Article Title: Characterization of Entry and Infection of Monocytic THP-1 cells by Kaposi's Sarcoma Associated Herpesvirus (KSHV): Role of Heparan Sulfate, DC-SIGN, Integrins and Signaling

doi: 10.1016/j.virol.2010.07.012

Figure Lengend Snippet: THP-1 cells were infected with 10 KSHV DNA copies per cell for 5 and 10 min. The cells were fixed in 2 % paraformaldehyde for 10 min, permeabilized with 0.2% Triton X-100 for 5 min and blocked. Infected and uninfected cells were stained with anti-KSHV gB and anti-α3β1/αvβ3/αvβ5/α5β1/αvβ6 integrin antibodies and visualized by confocal microscopy. KSHV-gB and integrins were visualized by incubation with Alexa-488 secondary antibody (green) and Alexa-594 secondary antibody (red), respectively. The arrows indicate the areas of KSHV co-localization with integrins. A. KSHV+ α3β1; B. KSHV+ αvβ3; C. KSHV+ αvβ5; D. KSHV+ α5β1; E. KSHV+ αvβ6.

Article Snippet: Rabbit anti-integrin α3 and β1 and soluble human integrins α3β1, αvβ3, αvβ5 and α5β1 were from Upstate Biotechnology, Lake Placid, N.Y. Recombinant human integrin α4β7 was from R&D systems, Minneapolis, MN.

Techniques: Infection, Staining, Confocal Microscopy, Incubation