Journal: eLife

Article Title: Matrix-regulated integrin α v β 5 maintains α 5 β 1 -dependent desmoplastic traits prognostic of neoplastic recurrence

doi: 10.7554/eLife.20600

Figure Lengend Snippet: ( A ) An integrin-dependent cell adhesion array test was used to assess the PM expression of integrin heterodimers in primary fibroblasts isolated from normal (white bars) vs. matched tumor tissue (desmoplastic; dark bars). Note that no differences were apparent between the two cell types with regards to levels of α v β 5 and α 5 β 1 integrins. ( B ) Naïve human pancreatic fibroblastic stellate cells were re-plated onto D-ECMs overnight in the presence of functional blocking anti-α v β 5 -integrin (ALULA ; α v β 5 -i), functional blocking anti-α 5 β 1 -integrin (mAb16 ; α 5 β 1 -i), combinations of both functional blocking antibodies (β 5 -i + α 5 -i), functional stabilizing anti-α 5 β 1 -integrin (SNAKA51 ; α 5 β 1 -act), or non-immunized isotypic antibodies (IgG). Representative monochromatic images of αSMA- and F-actin stained fibroblasts are shown. ( C ) Quantification of the experiment performed in ( B ). ( D ) Pseudocolored images depicting the intensity of αSMA expression including a color bar scale (0–255 intensity tone values). ( E ) Quantification of ( D ). Note that corresponding quantifications and p-values, for results shown in ( B–E ) are summarized in . ( F ) Naïve murine skin fibroblasts were re-plated onto murine D-ECMs (mD-ECM) produced by murine skin squamous cell carcinoma associated CAFs , and subjected to α v β 5 -integrin and α 5 β 1 -integrin inhibitors alone (ALULA: α v β 5 -i and BMA5: α 5 β 1 i) or in combination (β 5 + α 5 -i). The effects on myofibroblastic activation were measured for αSMA as in ( B ). The red asterisk illustrates the area outlined in red in the magnified insert for the intact (untreated) control. The same magnification is shown for the experimental conditions in the additional panels. As a method of quantifying the percentage of cells showing myofibroblastic features, the percentage of cells that have a stress fiber localized (αSMA) phenotype is shown (****p<0.0001). Note that inhibition of α 5 β 1 -integrin effectively reinstituted the mD-ECM-induced phenotype that was lost by inhibition of α v β 5 -integrin, just as seen above for the human PDAC system. Checkmarks identify conditions that resulted in myofibroblastic activation, while Xs identify conditions that did not result in myofibroblastic activation. ( G ) Model of D-ECM-induced activation of naïve fibroblasts, dependent on the activity of integrins α v β 5 and α 5 β 1 . Inhibition of α v β 5- integrin results in release of active α 5 β 1 -integrin, leading to blockade of D-ECM-induced myofibroblastic activation (1 st arrow, red X). The activity of α v β 5- integrin is no longer needed in the absence of α 5 β 1 -integrin activity, suggesting that α 5 β 1 -integrin activity in not necessary for fibroblasts to undergo D-ECM-induced myofibroblastic activation (2 nd arrow, green checkmark). Double inhibition of α v β 5 -integrin and α 5 β 1 -integrin results in D-ECM myofibroblastic activation, which proposes that inhibition of α 5 β 1 -integrin can overcome or rescue the effects seen under α v β 5 -integrin inhibition (3 rd arrow, green checkmark). Stabilization of α 5 β 1 -integrin in its active conformation overcomes the inhibitory/regulatory effects imparted by α v β 5 -integrin, resulting in ineffective D-ECM-induced myofibroblastic activation (4 th arrow, red X). Overall, the model suggests that D-ECM induces α v β 5 - integrin activity, which in turn results in the regulation of active α 5 β 1 -integrin, allowing D-ECM-induced myofibroblastic activation (large arrow to the right, green checkmark). DOI: http://dx.doi.org/10.7554/eLife.20600.015

Article Snippet: Membranes were then incubated in the one of the following primary antibodies overnight at 4°C: rabbit polyclonal anti-α v -integrin (AB_2631308) (1:5000) (Mybiosource, San Diego, CA), rabbit monoclonal anti-α 5 -integrin (AB_2631309) (1:1000) (Abcam, Cambridge, MA), rabbit polyclonal anti-β 5 -integrin (AB_10806204) (1:1000)(Millipore, Billerica, MA) or rabbit polyclonal anti-β 3 -integrin (1:1000) (AB_91119) (Millipore, Billerica, MA).

Techniques: Expressing, Isolation, Functional Assay, Blocking Assay, Staining, Produced, Activation Assay, Control, Inhibition, Activity Assay

Journal: eLife

Article Title: Matrix-regulated integrin α v β 5 maintains α 5 β 1 -dependent desmoplastic traits prognostic of neoplastic recurrence

doi: 10.7554/eLife.20600

Figure Lengend Snippet: αSMA stress fiber localization and expression levels in naïve fibroblasts (stellate cells) cultured overnight within D-ECMs in the presence of integrin functional antibodies. DOI: http://dx.doi.org/10.7554/eLife.20600.014

Article Snippet: Membranes were then incubated in the one of the following primary antibodies overnight at 4°C: rabbit polyclonal anti-α v -integrin (AB_2631308) (1:5000) (Mybiosource, San Diego, CA), rabbit monoclonal anti-α 5 -integrin (AB_2631309) (1:1000) (Abcam, Cambridge, MA), rabbit polyclonal anti-β 5 -integrin (AB_10806204) (1:1000)(Millipore, Billerica, MA) or rabbit polyclonal anti-β 3 -integrin (1:1000) (AB_91119) (Millipore, Billerica, MA).

Techniques: Expressing, Cell Culture, Functional Assay

Journal: eLife

Article Title: Matrix-regulated integrin α v β 5 maintains α 5 β 1 -dependent desmoplastic traits prognostic of neoplastic recurrence

doi: 10.7554/eLife.20600

Figure Lengend Snippet: ( A ) The CRISPR/CAS9 system was used to generate naïve fibroblastic stellate cell β 5 -integrin knock-out (KO) (β 5 -KO) or control KO (cntrl-KO) lines, whose genoype was confirmed by western blotting. The naïve cntrl-KO and β 5 -KO1 fibroblastic stellate cells were challenged by overnight culture within intact D-ECM and their myofibroblastic features were assessed. ( B ) Samples were subjected to indirect immunofluorescent labeling of αSMA and counterstained with fluorescently labeled phalloidin to detect actin stress fibers (F-actin) and representative monochromatic images are displayed. ( C ) Quantification of αSMA localized at actin stress fibers (F-Actin) from the experiment in ( B ) (***p=0.0006; n = 46). ( D ) Pseudocolored images represent intensity map outputs of αSMA, with an intensity color bar scale (0–255 intensity tone values) shown to the right. ( E ) Measured values from ( D ) are summarized in the graph (***p=0.0001; n = 46). An experimental condition consisting of naïve cntrl-KO fibroblastic stellate cells cultured in intact D-ECM was included in all experiments; this condition is summarized in this figure and is used for normalization (one arbitrary unit; a.u.). Checkmarks indicate conditions that induce a myofibroblastic activation phenotype, while Xs indicate loss of D-ECM-induced activation. DOI: http://dx.doi.org/10.7554/eLife.20600.016

Article Snippet: Membranes were then incubated in the one of the following primary antibodies overnight at 4°C: rabbit polyclonal anti-α v -integrin (AB_2631308) (1:5000) (Mybiosource, San Diego, CA), rabbit monoclonal anti-α 5 -integrin (AB_2631309) (1:1000) (Abcam, Cambridge, MA), rabbit polyclonal anti-β 5 -integrin (AB_10806204) (1:1000)(Millipore, Billerica, MA) or rabbit polyclonal anti-β 3 -integrin (1:1000) (AB_91119) (Millipore, Billerica, MA).

Techniques: CRISPR, Knock-Out, Control, Western Blot, Labeling, Cell Culture, Activation Assay

Journal: eLife

Article Title: Matrix-regulated integrin α v β 5 maintains α 5 β 1 -dependent desmoplastic traits prognostic of neoplastic recurrence

doi: 10.7554/eLife.20600

Figure Lengend Snippet: ( A ) The CRISPR/CAS9 system was used to generate α 5 -integrin knock-out (α 5 -KO), α v -integrin knock-out (α V -KO), β 3 -integrin knock-out (β 3 -KO) and control KO (cntrl-KO) naïve fibroblasts, which were confirmed by western blotting. The naïve cntrl-KO and assorted integrin KO fibroblastic stellate cells were challenged by overnight culture within intact D-ECM, and their myofibroblastic features were assessed. ( B ) All samples were subjected to indirect immunofluorescent labeling of αSMA and counterstained with fluorescently labeled phalloidin to detect actin stress fibers (F-actin) and representative monochromatic images are displayed. ( C ) Quantification of αSMA localized at actin stress fibers (F-actin) from the experiment in ( B ) (***p=0.0002; ****p<0.0001). ( D ) Pseudocolored images represent intensity maps of αSMA, with an intensity color bar scale (0–255 intensity tone values) shown to the right. ( E ) Measured values from ( D ) are summarized in the graph (****p<0.0001). An experimental condition consisting of naïve cntrl-KO fibroblastic stellate cells cultured in intact D-ECM was included in all experiments summarized in this figure and is used for normalization (one arbitrary unit; a.u., indicated by dotted lines in graphs). X marks indicate loss of D-ECM-induced activation for all tested mutant cells. DOI: http://dx.doi.org/10.7554/eLife.20600.017

Article Snippet: Membranes were then incubated in the one of the following primary antibodies overnight at 4°C: rabbit polyclonal anti-α v -integrin (AB_2631308) (1:5000) (Mybiosource, San Diego, CA), rabbit monoclonal anti-α 5 -integrin (AB_2631309) (1:1000) (Abcam, Cambridge, MA), rabbit polyclonal anti-β 5 -integrin (AB_10806204) (1:1000)(Millipore, Billerica, MA) or rabbit polyclonal anti-β 3 -integrin (1:1000) (AB_91119) (Millipore, Billerica, MA).

Techniques: CRISPR, Knock-Out, Control, Western Blot, Labeling, Cell Culture, Activation Assay, Mutagenesis

Journal: eLife

Article Title: Matrix-regulated integrin α v β 5 maintains α 5 β 1 -dependent desmoplastic traits prognostic of neoplastic recurrence

doi: 10.7554/eLife.20600

Figure Lengend Snippet: ( A ) Representative indirect immunofluorescent images of 3D D-ECM producing CAFs in the presence of functional blocking anti-α v β 5 -integrin (ALULA ; D + α v β 5 -i), active conformation stabilizing anti-α 5 β 1 -integrin (SNAKA51 ; D + α 5 β 1 -act.) or non-immunized isotypic antibodies (D + IgG). Spinning disk confocal monochromatic images, obtained following indirect immunofluorescence, show nuclei (Hoechst; yellow), αSMA (white) and ECM (fibronectin; magenta). ( B ) The corresponding ECM fiber angle distributions, determined by Image-J’s ‘Orientation J’ plugin, were normalized using hue values for a cyan mode angle visualization as shown in the bar on the right. ( C ) Corresponding curves depicting experimental-repetition-averaged variations of angle distributions normalized to 0° modes and summarizing the results. Dotted lines correspond to 15° fiber angle spreads. ( D ) Plotted data depicting summarized percentages of fibers distributed at 15° angles from the mode for each experimental condition. The dotted line denotes 55% alignment. Note how none of the treatments seem to have altered the myofibroblastic features of CAFs or their capability to produce anisotropic D-ECMs. DOI: http://dx.doi.org/10.7554/eLife.20600.018

Article Snippet: Membranes were then incubated in the one of the following primary antibodies overnight at 4°C: rabbit polyclonal anti-α v -integrin (AB_2631308) (1:5000) (Mybiosource, San Diego, CA), rabbit monoclonal anti-α 5 -integrin (AB_2631309) (1:1000) (Abcam, Cambridge, MA), rabbit polyclonal anti-β 5 -integrin (AB_10806204) (1:1000)(Millipore, Billerica, MA) or rabbit polyclonal anti-β 3 -integrin (1:1000) (AB_91119) (Millipore, Billerica, MA).

Techniques: Functional Assay, Blocking Assay, Immunofluorescence

Journal: eLife

Article Title: Matrix-regulated integrin α v β 5 maintains α 5 β 1 -dependent desmoplastic traits prognostic of neoplastic recurrence

doi: 10.7554/eLife.20600

Figure Lengend Snippet: Naïve fibroblasts were plated onto D-ECMs that were produced in the presence of functional blocking anti-α v β 5 -integrin (ALULA ; D + α v β 5 -i), active conformation stabilizing anti-α 5 β 1 -integrin (SNAKA51 ; D + α 5 β 1 -act.) or non-immunized isotypic antibodies (D + IgG), and αSMA and actin stress fibers (F-actin) were immunofluorescently labeled. ( A ) Monochromatic images indicative of double-labeled αSMA and F-actin are shown, while levels of total αSMA localized at corresponding stress fibers are plotted in the graph to the right. ( B ) Pseudocolored images represent αSMA intensity values, which are summarized in the graph to the right. Checkmarks indicate conditions that induce a myofibroblastic activation phenotype. Note that D-ECMs that were produced under α v β 5 -integrin inhibitory or active α 5 β 1 -integrin stabilizing conditions resulted in no apparent alteration of D-ECM function of CAFs. DOI: http://dx.doi.org/10.7554/eLife.20600.019

Article Snippet: Membranes were then incubated in the one of the following primary antibodies overnight at 4°C: rabbit polyclonal anti-α v -integrin (AB_2631308) (1:5000) (Mybiosource, San Diego, CA), rabbit monoclonal anti-α 5 -integrin (AB_2631309) (1:1000) (Abcam, Cambridge, MA), rabbit polyclonal anti-β 5 -integrin (AB_10806204) (1:1000)(Millipore, Billerica, MA) or rabbit polyclonal anti-β 3 -integrin (1:1000) (AB_91119) (Millipore, Billerica, MA).

Techniques: Produced, Functional Assay, Blocking Assay, Labeling, Activation Assay

Journal: eLife

Article Title: Matrix-regulated integrin α v β 5 maintains α 5 β 1 -dependent desmoplastic traits prognostic of neoplastic recurrence

doi: 10.7554/eLife.20600

Figure Lengend Snippet: Representative western blot of β5-integrin ( A ) and α 5 -integrin ( C ) expression in desmoplastic fibroblasts (CAFs), illustrating the result of CRISPR/CAS9-mediated KO of integrins (A — β5-KO1 + β5-KO2 and C — α 5 -integrin α 5 -KO1 + α 5 -KO2) compared to non-targeting gRNA control (cntrl-KO). Histone three was used as a loading control. Representative confocal microscopy images of either control CAF-KO (cntrl-KO) or CAF-β5-integrin-KO2 (β5-KO2) ( B ) or CAF-α 5 -integrin-KO2 (α 5 -KO2) ( D ), depicting αSMA (white) and nuclei (yellow). Inserts show the image-matching ECM fibers (fibronectin, magenta). The images on the right show the corresponding ECM fiber angle distributions as obtained using the Image-J’s ‘OrientationJ’ plug. ( E ) Quantification of the distribution of fiber angles that are within 15° of the mode from ( B ) (****p<0.0001). Note that α 5 -KO2 CAFs did not produce matrices that were substantial enough for quantification and were therefore omitted from ( E ). DOI: http://dx.doi.org/10.7554/eLife.20600.020

Article Snippet: Membranes were then incubated in the one of the following primary antibodies overnight at 4°C: rabbit polyclonal anti-α v -integrin (AB_2631308) (1:5000) (Mybiosource, San Diego, CA), rabbit monoclonal anti-α 5 -integrin (AB_2631309) (1:1000) (Abcam, Cambridge, MA), rabbit polyclonal anti-β 5 -integrin (AB_10806204) (1:1000)(Millipore, Billerica, MA) or rabbit polyclonal anti-β 3 -integrin (1:1000) (AB_91119) (Millipore, Billerica, MA).

Techniques: Western Blot, Expressing, CRISPR, Control, Confocal Microscopy

Journal: eLife

Article Title: Matrix-regulated integrin α v β 5 maintains α 5 β 1 -dependent desmoplastic traits prognostic of neoplastic recurrence

doi: 10.7554/eLife.20600

Figure Lengend Snippet: Representative western blots of α V -integrin ( A ) and β3-integrin ( C ) expression in CAFs, illustrating the result of CRISPR/CAS9 editing of α V -integrin (α V -KO1 + α V -KO2) ( A ) and β3-integrin (β3-KO1 + β3-KO2) ( C ) compared to non-targeting, gRNA, control (cntrl-KO). Histone three was used as a loading control. Representative confocal microscopy images of CAF-α V -integrin-KO1 (α V -KO1) ( B ) and CAF-β3-integrin-KO1 (β3-KO2) ( D ) depicting αSMA (white) or nuclei (yellow). Inserts show the image-matching ECM fibers (fibronectin, magenta). The images on the right are the corresponding ECMs, which for α V -KO1 was not substantial enough to pass the quality thickness test and was therefore omitted from further assessment; for β 3 -KO2, ECM fiber angle distributions shown by pseudocoloring were obtained using Image-J’s ‘OrientationJ’ plug in. ( E ) Quantification of the distribution of fiber angles that are within 15° of the mode shown in ( D ). The dotted line corresponds to the percentage of fiber alignment seen in control KO. DOI: http://dx.doi.org/10.7554/eLife.20600.021

Article Snippet: Membranes were then incubated in the one of the following primary antibodies overnight at 4°C: rabbit polyclonal anti-α v -integrin (AB_2631308) (1:5000) (Mybiosource, San Diego, CA), rabbit monoclonal anti-α 5 -integrin (AB_2631309) (1:1000) (Abcam, Cambridge, MA), rabbit polyclonal anti-β 5 -integrin (AB_10806204) (1:1000)(Millipore, Billerica, MA) or rabbit polyclonal anti-β 3 -integrin (1:1000) (AB_91119) (Millipore, Billerica, MA).

Techniques: Western Blot, Expressing, CRISPR, Control, Confocal Microscopy

Journal: eLife

Article Title: Matrix-regulated integrin α v β 5 maintains α 5 β 1 -dependent desmoplastic traits prognostic of neoplastic recurrence

doi: 10.7554/eLife.20600

Figure Lengend Snippet: Naïve fibroblasts were re-plated onto D-ECMs and challenged with either control conditions (DMSO + IgG), small molecule FAK inhibitor PF573,228 (FAK-i) alone or FAK inhibitor in combination with α 5 β 1 -integrin inhibitor (FAK-i + α 5 -i, mAb16 ), and activation of fibroblasts was tested. ( A ) Representative monochromatic images of immunofluorescently labeled αSMA and actin stress fibers (F-actin). Colored asterisks in ( A ) represent areas that are magnified in the corresponding panels to the right. ( B ) Quantification of αSMA at actin stress fibers (F-actin) from the experiment in ( A ) and normalized to DMSO + IgG control (one arbitrary unit; a.u.) (****p<0.0001). ( C ) Pseudocolored images represent intensity maps of αSMA, with an intensity color bar scale (0–255 intensity tone values) shown to the right. ( D ) Quantification of αSMA intensity from ( C ) (***p=0.0001). Note that the D-ECM-induced phenotype that was lost under FAK inhibition was rescued under α 5 β 1 -integrin co-inhibition (just as for the two integrin co-inhibitions shown in ). DOI: http://dx.doi.org/10.7554/eLife.20600.022

Article Snippet: Membranes were then incubated in the one of the following primary antibodies overnight at 4°C: rabbit polyclonal anti-α v -integrin (AB_2631308) (1:5000) (Mybiosource, San Diego, CA), rabbit monoclonal anti-α 5 -integrin (AB_2631309) (1:1000) (Abcam, Cambridge, MA), rabbit polyclonal anti-β 5 -integrin (AB_10806204) (1:1000)(Millipore, Billerica, MA) or rabbit polyclonal anti-β 3 -integrin (1:1000) (AB_91119) (Millipore, Billerica, MA).

Techniques: Control, Activation Assay, Labeling, Inhibition

Journal: eLife

Article Title: Matrix-regulated integrin α v β 5 maintains α 5 β 1 -dependent desmoplastic traits prognostic of neoplastic recurrence

doi: 10.7554/eLife.20600

Figure Lengend Snippet: Naïve murine skin fibroblasts were re-plated onto murine D-ECMs (mD-ECM) and were treated with PF573,228 (FAK inhibitor) and also subjected to α v β 5 -integrin or α 5 β 1 -integrin inhibitors (ALULA — α v β 5 -i or BMA5 — α 5 β 1 i) or IgG control. The effects on naïve-to-myofibroblastic activation were measured in all treated PF573,228 conditions by indirect immunofluorescence of αSMA expression. ( A ) Representative monochromatic images of stress fiber localized αSMA. ( B ) Quantification of the percentage of cells showing myofibroblastic features (stress fiber localized αSMA phenotype %) during the experiment illustrated in ( A ) (***p=0.0001). Note that co-inhibition of FAK and α 5 β 1 -integrin effectively reinstituted the mD-ECM-induced phenotype lost as a result of FAK inhibition alone (cnt), just as in the human PDAC stroma system and as was the case for the two integrin co-inhibitions shown in . DOI: http://dx.doi.org/10.7554/eLife.20600.023

Article Snippet: Membranes were then incubated in the one of the following primary antibodies overnight at 4°C: rabbit polyclonal anti-α v -integrin (AB_2631308) (1:5000) (Mybiosource, San Diego, CA), rabbit monoclonal anti-α 5 -integrin (AB_2631309) (1:1000) (Abcam, Cambridge, MA), rabbit polyclonal anti-β 5 -integrin (AB_10806204) (1:1000)(Millipore, Billerica, MA) or rabbit polyclonal anti-β 3 -integrin (1:1000) (AB_91119) (Millipore, Billerica, MA).

Techniques: Control, Activation Assay, Immunofluorescence, Expressing, Inhibition

Journal: eLife

Article Title: Matrix-regulated integrin α v β 5 maintains α 5 β 1 -dependent desmoplastic traits prognostic of neoplastic recurrence

doi: 10.7554/eLife.20600

Figure Lengend Snippet: FAK null (FAK -/-) naïve murine skin fibroblasts were re-plated onto murine D-ECMs (mD-ECM) subjected to α v β 5 -integrin and α 5 β 1 -integrin inhibitors (ALULA — α v β 5 -i or BMA5 — α 5 β 1 i). The effects on myofibroblastic activation were measured by indirect immunofluorescence of αSMA expression as before. ( A ) Representative monochromatic images of αSMA expression. ( B ) Quantification of the percentage of cells showing myofibroblastic features (stress fiber localized αSMA phenotype %) during the experiment illustrated in ( A ) (****p<0.0001). Note that inhibition of α 5 β 1 -integrin effectively reinstituted the mD-ECM-induced phenotype that was lost as a result of FAK deletion. DOI: http://dx.doi.org/10.7554/eLife.20600.024

Article Snippet: Membranes were then incubated in the one of the following primary antibodies overnight at 4°C: rabbit polyclonal anti-α v -integrin (AB_2631308) (1:5000) (Mybiosource, San Diego, CA), rabbit monoclonal anti-α 5 -integrin (AB_2631309) (1:1000) (Abcam, Cambridge, MA), rabbit polyclonal anti-β 5 -integrin (AB_10806204) (1:1000)(Millipore, Billerica, MA) or rabbit polyclonal anti-β 3 -integrin (1:1000) (AB_91119) (Millipore, Billerica, MA).

Techniques: Activation Assay, Immunofluorescence, Expressing, Inhibition

Journal: eLife

Article Title: Matrix-regulated integrin α v β 5 maintains α 5 β 1 -dependent desmoplastic traits prognostic of neoplastic recurrence

doi: 10.7554/eLife.20600

Figure Lengend Snippet: Wild-type (WT) or FAK kinase dead (KD) naïve murine skin fibroblasts were re-plated onto murine D-ECMs (mD-ECM) and subjected to α 5 β 1 -integrin inhibitor (BMA5 — WT α 5 i or KD α 5 i) or IgG control (WT cnt and KD IgG)). The effects on myofibroblastic activation were measured by indirect immunofluorescence of αSMA expression. ( A ) Representative monochromatic images of αSMA expression. ( B ) Quantification of the percentage of cells showing myofibroblastic features (stress fiber localized αSMA phenotype %) during the experiment illustrated in ( A ). Note that inhibition of α 5 β 1 -integrin effectively reinstituted the mD-ECM-induced phenotype that was lost as a result of FAK KD mutation. DOI: http://dx.doi.org/10.7554/eLife.20600.025

Article Snippet: Membranes were then incubated in the one of the following primary antibodies overnight at 4°C: rabbit polyclonal anti-α v -integrin (AB_2631308) (1:5000) (Mybiosource, San Diego, CA), rabbit monoclonal anti-α 5 -integrin (AB_2631309) (1:1000) (Abcam, Cambridge, MA), rabbit polyclonal anti-β 5 -integrin (AB_10806204) (1:1000)(Millipore, Billerica, MA) or rabbit polyclonal anti-β 3 -integrin (1:1000) (AB_91119) (Millipore, Billerica, MA).

Techniques: Control, Activation Assay, Immunofluorescence, Expressing, Inhibition, Mutagenesis

Journal: eLife

Article Title: Matrix-regulated integrin α v β 5 maintains α 5 β 1 -dependent desmoplastic traits prognostic of neoplastic recurrence

doi: 10.7554/eLife.20600

Figure Lengend Snippet: SRC naïve murine skin fibroblasts lacking SRC, YES and FYN (SYF -/-) were re-plated onto murine D-ECMs (mD-ECM) and α v β 5 -integrin and α 5 β 1 -integrin inhibitors (ALULA — α v β 5 -i and BMA5 — α 5 β 1 i) or IgG control (IgG). The effects on myofibroblastic activation were measured by indirect immunofluorescence of αSMA expression. ( A ) Representative monochromatic images of αSMA expression. ( B ) Quantification of the percentage of cells showing myofibroblastic features (stress fiber localized αSMA phenotype %) during the experiment illustrated in ( A ). Note that inhibition of α 5 β 1 -integrin could not reinstitute the mD-ECM-induced phenotype that was lost in SRC -/- cells. DOI: http://dx.doi.org/10.7554/eLife.20600.026

Article Snippet: Membranes were then incubated in the one of the following primary antibodies overnight at 4°C: rabbit polyclonal anti-α v -integrin (AB_2631308) (1:5000) (Mybiosource, San Diego, CA), rabbit monoclonal anti-α 5 -integrin (AB_2631309) (1:1000) (Abcam, Cambridge, MA), rabbit polyclonal anti-β 5 -integrin (AB_10806204) (1:1000)(Millipore, Billerica, MA) or rabbit polyclonal anti-β 3 -integrin (1:1000) (AB_91119) (Millipore, Billerica, MA).

Techniques: Control, Activation Assay, Immunofluorescence, Expressing, Inhibition

Journal: eLife

Article Title: Matrix-regulated integrin α v β 5 maintains α 5 β 1 -dependent desmoplastic traits prognostic of neoplastic recurrence

doi: 10.7554/eLife.20600

Figure Lengend Snippet: Naïve fibroblastic stellate control KO (cntrl-KO) or α 5 -integrin (α 5 -KO) cells were re-plated within intact D-ECMs and challenged with DMSO as control or with the small molecule FAK inhibitor PF573,228 (FAK-i), and the activation of fibroblasts was tested. Representative monochromatic images of αSMA and actin stress fibers (F-actin) were immunofluorescently labeled and are shown. Note that unlike α 5 β 1 -integrin inhibition with mAb16, loss of α 5 -integrin expression did not rescue the myofibroblastic phenotype. DOI: http://dx.doi.org/10.7554/eLife.20600.027

Article Snippet: Membranes were then incubated in the one of the following primary antibodies overnight at 4°C: rabbit polyclonal anti-α v -integrin (AB_2631308) (1:5000) (Mybiosource, San Diego, CA), rabbit monoclonal anti-α 5 -integrin (AB_2631309) (1:1000) (Abcam, Cambridge, MA), rabbit polyclonal anti-β 5 -integrin (AB_10806204) (1:1000)(Millipore, Billerica, MA) or rabbit polyclonal anti-β 3 -integrin (1:1000) (AB_91119) (Millipore, Billerica, MA).

Techniques: Control, Activation Assay, Labeling, Inhibition, Expressing

Journal: eLife

Article Title: Matrix-regulated integrin α v β 5 maintains α 5 β 1 -dependent desmoplastic traits prognostic of neoplastic recurrence

doi: 10.7554/eLife.20600

Figure Lengend Snippet: ( A ) Indirect immunofluorescent and spinning disc confocal generated images of 3D-adhesions (identified using mAb11) , formed by naïve fibroblastic cells cultured within N-ECM or D-ECM in the absence (cnt.) or presence of ALULA for α v β 5 -integrin inhibition (α v β 5 -i) or SNAKA52 to stabilize α 5 β 1 -integrin activity (α 5 β 1 act) or IgG as control. (A) The artificially colored structures represent computer-selected internally threshold objects (ITOs) of 3D-adhesion structures. ( B ) Quantification of the length of ITO generated objects from ( A ) (***p=0.0026. ****p<0.0001). Note the significant differences in 3D-adhesion length observed between N-ECM and D-ECM as well as between IgG and SNAKA51 treatments. DOI: http://dx.doi.org/10.7554/eLife.20600.028

Article Snippet: Membranes were then incubated in the one of the following primary antibodies overnight at 4°C: rabbit polyclonal anti-α v -integrin (AB_2631308) (1:5000) (Mybiosource, San Diego, CA), rabbit monoclonal anti-α 5 -integrin (AB_2631309) (1:1000) (Abcam, Cambridge, MA), rabbit polyclonal anti-β 5 -integrin (AB_10806204) (1:1000)(Millipore, Billerica, MA) or rabbit polyclonal anti-β 3 -integrin (1:1000) (AB_91119) (Millipore, Billerica, MA).

Techniques: Generated, Cell Culture, Inhibition, Activity Assay, Control

Journal: eLife

Article Title: Matrix-regulated integrin α v β 5 maintains α 5 β 1 -dependent desmoplastic traits prognostic of neoplastic recurrence

doi: 10.7554/eLife.20600

Figure Lengend Snippet: Summary of results from naïve fibroblasts cultured within assorted ECMs in the absence (N-ECM or D-ECM) or presence of α v β 5 -integrin inhibitor ALULA (D-ECM + α v β 5 -i) and stained for adhesion structures and active α 5 β 1 -integrin. The sizes of pie graphs are relative to SMIA-CUKIE output corresponding to total intensity levels, while relative percentage distributions at 3D-structures (yellow) and away from these structures (light green) are shown. Note that the percentage distributions between locations at and away from 3D-adhesions are relatively unchanged while total intensity levels of active integrin are increased in response to D-ECM. DOI: http://dx.doi.org/10.7554/eLife.20600.031

Article Snippet: Membranes were then incubated in the one of the following primary antibodies overnight at 4°C: rabbit polyclonal anti-α v -integrin (AB_2631308) (1:5000) (Mybiosource, San Diego, CA), rabbit monoclonal anti-α 5 -integrin (AB_2631309) (1:1000) (Abcam, Cambridge, MA), rabbit polyclonal anti-β 5 -integrin (AB_10806204) (1:1000)(Millipore, Billerica, MA) or rabbit polyclonal anti-β 3 -integrin (1:1000) (AB_91119) (Millipore, Billerica, MA).

Techniques: Cell Culture, Staining

Journal: eLife

Article Title: Matrix-regulated integrin α v β 5 maintains α 5 β 1 -dependent desmoplastic traits prognostic of neoplastic recurrence

doi: 10.7554/eLife.20600

Figure Lengend Snippet: Naïve cells were plated overnight within D-ECM. ( A ) Indirect immunofluorescent images of active α 5 β 1 -integrin (α 5 β 1 -act.; SNAKA51 in green) locations relative to 3D-adhesions (3D-adh.; mAb11 in red), showing a representative cell under permeable vs. non-permeable conditions. The third image (on the right)demonstrates the specificity of active α 5 β 1 -integrin detection when samples were treated with functional blocking anti-α 5 β 1 -integrin antibody, mAb16, under permeable conditions (perm. + α 5 β 1 i). ( B ) Graph summarizes permeable vs. non-permeable values of active α 5 β 1 -integrin from experimental repetitions in which median permeable intensity levels were used for normalization (one arbitrary unit; a.u.) (***p=0.0005). DOI: http://dx.doi.org/10.7554/eLife.20600.032

Article Snippet: Membranes were then incubated in the one of the following primary antibodies overnight at 4°C: rabbit polyclonal anti-α v -integrin (AB_2631308) (1:5000) (Mybiosource, San Diego, CA), rabbit monoclonal anti-α 5 -integrin (AB_2631309) (1:1000) (Abcam, Cambridge, MA), rabbit polyclonal anti-β 5 -integrin (AB_10806204) (1:1000)(Millipore, Billerica, MA) or rabbit polyclonal anti-β 3 -integrin (1:1000) (AB_91119) (Millipore, Billerica, MA).

Techniques: Functional Assay, Blocking Assay

Journal: eLife

Article Title: Matrix-regulated integrin α v β 5 maintains α 5 β 1 -dependent desmoplastic traits prognostic of neoplastic recurrence

doi: 10.7554/eLife.20600

Figure Lengend Snippet: ( A ) Double-labeled images depicting 3D-adhesion (red in overlay; 3D-adh.) and active α 5 β 1 -integrin (green in overlay; α 5 β 1 -act) in naïve fibroblasts cultured overnight in normal-ECMs (N-ECM) or desmoplastic-ECMs (D-ECM). The pseudocolored images on the far right represent semi-quantitative images of maximum reconstructions of active α 5 β 1 -integrin levels (α 5 β 1 -act inten), with a corresponding intensity bar shown on the right. ( B ) Summary of total active α 5 β 1 -integrin levels using median levels on D-ECM for normalization (one arbitrary unit; a.u.) (****p<0.0001). DOI: http://dx.doi.org/10.7554/eLife.20600.029

Article Snippet: Membranes were then incubated in the one of the following primary antibodies overnight at 4°C: rabbit polyclonal anti-α v -integrin (AB_2631308) (1:5000) (Mybiosource, San Diego, CA), rabbit monoclonal anti-α 5 -integrin (AB_2631309) (1:1000) (Abcam, Cambridge, MA), rabbit polyclonal anti-β 5 -integrin (AB_10806204) (1:1000)(Millipore, Billerica, MA) or rabbit polyclonal anti-β 3 -integrin (1:1000) (AB_91119) (Millipore, Billerica, MA).

Techniques: Labeling, Cell Culture

Journal: eLife

Article Title: Matrix-regulated integrin α v β 5 maintains α 5 β 1 -dependent desmoplastic traits prognostic of neoplastic recurrence

doi: 10.7554/eLife.20600

Figure Lengend Snippet: Naïve fibroblasts were cultured within assorted ECMs in the absence (N-ECM or D-ECM) or presence of α v β 5 -integrin inhibitor ALULA (D-ECM + α v β 5 -i) and were stained for adhesion structures, pFAK, and active α 5 β 1 -integrin and the images were quantified. ( A ) Graph depicting active α 5 β 1 -integrin levels localized at 3D-adhesions, normalized to D-ECM median intensities, calculated using SMIA-CUKIE publicly available at https://github.com/cukie/SMIA . ( B ) Graph depicting levels of pFAK-Y 397 , calculated using SMIA-CUKIE, localized at 3D-adhesions structures (****p<0.0001). ( C ) Graph showing active α 5 β 1 -integrin intensity values, normalized to D-ECM mean intensities and calculated using SMIA-CUKIE, that are localized away from 3D-adhesions (****p<0.0001). Note that the D-ECM-induced increases in active α 5 β 1 -integrin, which are evident at locations away from 3D-adhesions, are concomitant with increased 3D-adhesion localized pFAK. DOI: http://dx.doi.org/10.7554/eLife.20600.030

Article Snippet: Membranes were then incubated in the one of the following primary antibodies overnight at 4°C: rabbit polyclonal anti-α v -integrin (AB_2631308) (1:5000) (Mybiosource, San Diego, CA), rabbit monoclonal anti-α 5 -integrin (AB_2631309) (1:1000) (Abcam, Cambridge, MA), rabbit polyclonal anti-β 5 -integrin (AB_10806204) (1:1000)(Millipore, Billerica, MA) or rabbit polyclonal anti-β 3 -integrin (1:1000) (AB_91119) (Millipore, Billerica, MA).

Techniques: Cell Culture, Staining

Journal: eLife

Article Title: Matrix-regulated integrin α v β 5 maintains α 5 β 1 -dependent desmoplastic traits prognostic of neoplastic recurrence

doi: 10.7554/eLife.20600

Figure Lengend Snippet: Summary of results from naïve fibroblasts cultured within assorted ECMs in the absence (N-ECM or D-ECM) or presence of α v β 5 -integrin inhibitor ALULA (D-ECM + α v β 5 -i) and stained, following permeable vs. non-permeable conditions, for adhesion structures and active α 5 β 1 -integrin. The pie graphs depict active α 5 β 1 -integrin fractions localized exogenously on the PM at (non-permeable conditions; yellow or away from (light green) 3D-adhesions, or intracellularly (dark green). Diminished levels of intracellular active α 5 β 1 -integrin are represented by the ‘empty’ pie wedges (gray). Data were normalized to D-ECM-induced levels obtained under permeable conditions as in – . DOI: http://dx.doi.org/10.7554/eLife.20600.033

Article Snippet: Membranes were then incubated in the one of the following primary antibodies overnight at 4°C: rabbit polyclonal anti-α v -integrin (AB_2631308) (1:5000) (Mybiosource, San Diego, CA), rabbit monoclonal anti-α 5 -integrin (AB_2631309) (1:1000) (Abcam, Cambridge, MA), rabbit polyclonal anti-β 5 -integrin (AB_10806204) (1:1000)(Millipore, Billerica, MA) or rabbit polyclonal anti-β 3 -integrin (1:1000) (AB_91119) (Millipore, Billerica, MA).

Techniques: Cell Culture, Staining

Journal: eLife

Article Title: Matrix-regulated integrin α v β 5 maintains α 5 β 1 -dependent desmoplastic traits prognostic of neoplastic recurrence

doi: 10.7554/eLife.20600

Figure Lengend Snippet: ( A ) Pseudocolored images showing the intensities of representative indirect immunofluorescence images indicating active α 5 β 1 -integrin (α 5 β 1 -act.) or pFAK from control KO (cntrl KO) or β 5 -integrin KO (β 5 -KO1) naïve fibroblasts. The fibroblasts were cultured overnight within D-ECMs. Intensity scale bars are shown to the right. ( B and C ) Quantification of total active α 5 β 1 -integrin (**p=0.0420) ( B ) and pFAK-Y 397 (**p=0.0246) ( C ) levels of cells from ( A ). Note that both activities that were induced by D-ECM in naïve fibroblasts are lost in β 5 -integrin KO naïve fibroblastic stellate cells. DOI: http://dx.doi.org/10.7554/eLife.20600.034

Article Snippet: Membranes were then incubated in the one of the following primary antibodies overnight at 4°C: rabbit polyclonal anti-α v -integrin (AB_2631308) (1:5000) (Mybiosource, San Diego, CA), rabbit monoclonal anti-α 5 -integrin (AB_2631309) (1:1000) (Abcam, Cambridge, MA), rabbit polyclonal anti-β 5 -integrin (AB_10806204) (1:1000)(Millipore, Billerica, MA) or rabbit polyclonal anti-β 3 -integrin (1:1000) (AB_91119) (Millipore, Billerica, MA).

Techniques: Immunofluorescence, Control, Cell Culture

Journal: eLife

Article Title: Matrix-regulated integrin α v β 5 maintains α 5 β 1 -dependent desmoplastic traits prognostic of neoplastic recurrence

doi: 10.7554/eLife.20600

Figure Lengend Snippet: ( A ) Pseudocolored images showing the intensities of representative indirect immunofluorescence images indicating active α 5 β 1 -integrin (α 5 β 1 -act.) or pFAK from α 5 -integrin KO (α 5 -KO1), α V -integrin KO (α V -KO1) or β 3 -integrin KO (β 3 -KO1) naïve fibroblasts cultured overnight with D-ECMs. ( B ) Quantification of total active α 5 β 1 -integrin levels of cells from ( A ). ( C ) Quantifiction of pFAK levels of cells from ( A (**p=0.0343). Dotted lines in ( B ) and ( denote control KO normalized levels shown in . DOI: http://dx.doi.org/10.7554/eLife.20600.035

Article Snippet: Membranes were then incubated in the one of the following primary antibodies overnight at 4°C: rabbit polyclonal anti-α v -integrin (AB_2631308) (1:5000) (Mybiosource, San Diego, CA), rabbit monoclonal anti-α 5 -integrin (AB_2631309) (1:1000) (Abcam, Cambridge, MA), rabbit polyclonal anti-β 5 -integrin (AB_10806204) (1:1000)(Millipore, Billerica, MA) or rabbit polyclonal anti-β 3 -integrin (1:1000) (AB_91119) (Millipore, Billerica, MA).

Techniques: Immunofluorescence, Cell Culture, Control

Journal: eLife

Article Title: Matrix-regulated integrin α v β 5 maintains α 5 β 1 -dependent desmoplastic traits prognostic of neoplastic recurrence

doi: 10.7554/eLife.20600

Figure Lengend Snippet: ( A ) Representative indirect immunofluorescent images corresponding to overnight ‘chase’ incubations with pre-labeled anti-active-α 5 β 1 -integrin antibodies (SNAKA51 ) or IgG controls (blue, –not shown), followed by de novo detected active α 5 β 1 -integrin labeling after fixation (α 5 β 1 -act. in green) relative to 3D-adhesion structures (3D-adh. in red) under permeable vs. non-permeable conditions. Note how SNAKA51 treatment but not IgG prompts the relocation of integrin activity to the PM while there is practically no change between permeable and non-permeable active α 5 β 1 -integrin levels. ( B ) Transmitted electron microscopy images of double immunogold-labeled 3D-adhesions (–3D-adh. large particles, ) vs. active α 5 β 1 -integrin (-α 5 β 1 -act. small particles, ), detected in naïve cells cultured within N–ECM vs. D-ECM in the presence or absence of α v β 5 -integrin blockage using ALULA (αvβ5-i). Both reduction and relocation of active α 5 β 1 -integrin pools are observed. Arrowheads point at random immunogold particles as examples, while the closed arrow indicates the location of a clathrin-coated vesicle. DOI: http://dx.doi.org/10.7554/eLife.20600.036

Article Snippet: Membranes were then incubated in the one of the following primary antibodies overnight at 4°C: rabbit polyclonal anti-α v -integrin (AB_2631308) (1:5000) (Mybiosource, San Diego, CA), rabbit monoclonal anti-α 5 -integrin (AB_2631309) (1:1000) (Abcam, Cambridge, MA), rabbit polyclonal anti-β 5 -integrin (AB_10806204) (1:1000)(Millipore, Billerica, MA) or rabbit polyclonal anti-β 3 -integrin (1:1000) (AB_91119) (Millipore, Billerica, MA).

Techniques: Labeling, Activity Assay, Electron Microscopy, Cell Culture

Journal: eLife

Article Title: Matrix-regulated integrin α v β 5 maintains α 5 β 1 -dependent desmoplastic traits prognostic of neoplastic recurrence

doi: 10.7554/eLife.20600

Figure Lengend Snippet: Transmitted electron microscopy images of double immunogold-labeled 3D-adhesions (–3D-adh. large particles, ) vs. active α 5 β 1 -integrin (-α 5 β 1 -act. small particles, ), detected in naïve control KO (cntrl-KO) or β 5 -integrin KO (β 5 -KO1) fibroblastic stellate cells cultured within D-ECM. The images show a reduction of the total amounts of active α 5 β 1 -integrin and relocation of active α 5 β 1 -integrin pools to the PM in β 5 -integrin KO fibroblasts. The magnified insert depicts an example of intracellular pools of active α 5 β 1 -integrin at what appears to be multi-vesicular endosomes. Note that small particles, indicative of active α 5 β 1 -integrin, seemed to be absent from the clathrin-coated vesicle; these particles are indicated by the closed arrow, while arrowheads point to examples of random immunogold particles. DOI: http://dx.doi.org/10.7554/eLife.20600.037

Article Snippet: Membranes were then incubated in the one of the following primary antibodies overnight at 4°C: rabbit polyclonal anti-α v -integrin (AB_2631308) (1:5000) (Mybiosource, San Diego, CA), rabbit monoclonal anti-α 5 -integrin (AB_2631309) (1:1000) (Abcam, Cambridge, MA), rabbit polyclonal anti-β 5 -integrin (AB_10806204) (1:1000)(Millipore, Billerica, MA) or rabbit polyclonal anti-β 3 -integrin (1:1000) (AB_91119) (Millipore, Billerica, MA).

Techniques: Electron Microscopy, Labeling, Control, Cell Culture

Journal: eLife

Article Title: Matrix-regulated integrin α v β 5 maintains α 5 β 1 -dependent desmoplastic traits prognostic of neoplastic recurrence

doi: 10.7554/eLife.20600

Figure Lengend Snippet: Control KO (cntrl-KO) or β 5 -integrin knock-out (β 5 -KO1) naïve fibroblastic stellate cells were cultured overnight in desmoplastic-ECMs (D-ECM), and were subjected to indirect immunofluorescence using SNAKA51 to detect active α 5 β 1 -integrin (α 5 β 1 -act in green) in combination with one of the following endosomal markers shown in red: anti-EEA-1 (for early endosome), anti-Rab5 (for clathrin-mediated endocytosis early endosome), anti-Rab7 (late endosome to be degraded, recycled or rerouted), anti-Rab11 (late endosome to be recycled), or anti-CD81 (multivesicular endosomes). Top panels: confocal images were captured for each double-stained condition to identify the localization of active α 5 β 1 -integrin in relation to the assorted types of endosomes shown in red. Yellow arrowheads point to assorted endosomes and are identically placed in the slightly zoomed monochromatic inserts shown below, which allow better appreciation of the relative locations of the markers vs. those of active α 5 β 1 -integrin. Note that the partial co-localization of active α 5 β 1 -integrin with Rab7, Rab11 and especially with CD81 is lost in the naïve β 5 -KO compared to control KO naive fibroblasts in response to D-ECM. DOI: http://dx.doi.org/10.7554/eLife.20600.038

Article Snippet: Membranes were then incubated in the one of the following primary antibodies overnight at 4°C: rabbit polyclonal anti-α v -integrin (AB_2631308) (1:5000) (Mybiosource, San Diego, CA), rabbit monoclonal anti-α 5 -integrin (AB_2631309) (1:1000) (Abcam, Cambridge, MA), rabbit polyclonal anti-β 5 -integrin (AB_10806204) (1:1000)(Millipore, Billerica, MA) or rabbit polyclonal anti-β 3 -integrin (1:1000) (AB_91119) (Millipore, Billerica, MA).

Techniques: Control, Knock-Out, Cell Culture, Immunofluorescence, Staining

Journal: eLife

Article Title: Matrix-regulated integrin α v β 5 maintains α 5 β 1 -dependent desmoplastic traits prognostic of neoplastic recurrence

doi: 10.7554/eLife.20600

Figure Lengend Snippet: Representative indirect immunofluorescence images showing active α 5 β 1 -integrin (with SNAKA51 [α 5 β 1 -act.]) or pFAK in control KO (cntrl-KO) or β 5 -integrin KO (β5-KO1) CAFs at the completion of the 3D matrix production process (see Materials and methods). ( B - C ) Graphs depicting levels of active α 5 β 1 -integrin ( B ) or pFAK ( C ;**p=0.0465) in control and β 5 -integrin KO CAFs from ( A ). Note that active α 5 β 1 -integrin levels in CAFs were not significantly changed in response to β 5 -integrin loss. DOI: http://dx.doi.org/10.7554/eLife.20600.039

Article Snippet: Membranes were then incubated in the one of the following primary antibodies overnight at 4°C: rabbit polyclonal anti-α v -integrin (AB_2631308) (1:5000) (Mybiosource, San Diego, CA), rabbit monoclonal anti-α 5 -integrin (AB_2631309) (1:1000) (Abcam, Cambridge, MA), rabbit polyclonal anti-β 5 -integrin (AB_10806204) (1:1000)(Millipore, Billerica, MA) or rabbit polyclonal anti-β 3 -integrin (1:1000) (AB_91119) (Millipore, Billerica, MA).

Techniques: Immunofluorescence, Control

Journal: eLife

Article Title: Matrix-regulated integrin α v β 5 maintains α 5 β 1 -dependent desmoplastic traits prognostic of neoplastic recurrence

doi: 10.7554/eLife.20600

Figure Lengend Snippet: ( A ) Representative indirect immunofluorescence images showing active α 5 β 1 -integrin (with SNAKA51 [α 5 β 1 -act.]) or pFAK corresponding to α 5 -integrin KO (α 5 -KO2), α V -integrin KO (α V -KO1) or β 3 -integrin KO (β 3 -KO1) CAFs at the conclusion of matrix production (see Materials and methods). ( B - C ) Graphs depicting levels of active α 5 β 1 -integrin ( B ) or pFAK ( C ) in control and β 5 -integrin KO CAFs from ( A ) (****p<0.0001). Dotted lines in ( B – C ) denote control KO CAF normalized levels from . Note that active α 5 β 1 -integrin levels in CAFs were altered in response to β 3 - but not α V -integrin loss, whereas levels detected in α 5 -integrin KO CAFs served as background control and were therefore marked as ‘not applicable’ ( NA ). DOI: http://dx.doi.org/10.7554/eLife.20600.040

Article Snippet: Membranes were then incubated in the one of the following primary antibodies overnight at 4°C: rabbit polyclonal anti-α v -integrin (AB_2631308) (1:5000) (Mybiosource, San Diego, CA), rabbit monoclonal anti-α 5 -integrin (AB_2631309) (1:1000) (Abcam, Cambridge, MA), rabbit polyclonal anti-β 5 -integrin (AB_10806204) (1:1000)(Millipore, Billerica, MA) or rabbit polyclonal anti-β 3 -integrin (1:1000) (AB_91119) (Millipore, Billerica, MA).

Techniques: Immunofluorescence, Control

Journal: eLife

Article Title: Matrix-regulated integrin α v β 5 maintains α 5 β 1 -dependent desmoplastic traits prognostic of neoplastic recurrence

doi: 10.7554/eLife.20600

Figure Lengend Snippet: Fibroblasts were isolated from RCC surgical pathologically normal or tumoral samples, and their ECM-producing phenotypes were assessed after seven days of matrix production. ( A ) An integrin-dependent cell adhesion array test was used to assess the PM expression of integrin heterodimers in primary fibroblasts isolated from normal (white bars) vs. tumoral (dark bars) tissues. Note that no differences were apparent between the two cell types with regards to levels of α v β 5 and α 5 β 1 integrins. ( B ) Normal vs. desmoplastic mRNAs levels, corresponding to αSMA and palladin (used as an additional myofibroblastic marker as before) were obtained via RT-qPCR from the indicated 3D-cultures following renal ECM (rECM) production, which was achieved via confluent culturing of fibroblasts in the presence of ascorbic acid for a period lasting 8 days (**p=0.0286). ( C ) Representative images of normal vs. desmoplastic phenotypes, subsequent to 3D rECM production, are shown; comparison of low vs. high αSMA levels (white), heterogeneous/round vs. elongated/spindled nuclei (yellow) and disorganized/isotropic vs. parallel aligned/anisotropic rECMs (magenta) are evident in the representative images. DOI: http://dx.doi.org/10.7554/eLife.20600.041

Article Snippet: Membranes were then incubated in the one of the following primary antibodies overnight at 4°C: rabbit polyclonal anti-α v -integrin (AB_2631308) (1:5000) (Mybiosource, San Diego, CA), rabbit monoclonal anti-α 5 -integrin (AB_2631309) (1:1000) (Abcam, Cambridge, MA), rabbit polyclonal anti-β 5 -integrin (AB_10806204) (1:1000)(Millipore, Billerica, MA) or rabbit polyclonal anti-β 3 -integrin (1:1000) (AB_91119) (Millipore, Billerica, MA).

Techniques: Isolation, Expressing, Marker, Quantitative RT-PCR, Comparison

Journal: eLife

Article Title: Matrix-regulated integrin α v β 5 maintains α 5 β 1 -dependent desmoplastic traits prognostic of neoplastic recurrence

doi: 10.7554/eLife.20600

Figure Lengend Snippet: Naïve renal fibroblasts were cultured overnight within normal (rN-ECM) or RCC-associated CAF-derived ECMs (rD-ECM). rD-ECMs were produced in the presence of vehicle control (D + DMSO) or TGFβ1-receptor inhibitor (D + TGFβi). Alternatively, naïve cells cultured within rD-ECMs were treated with TGFβ1 inhibitor (TGFβ-i), with vehicle (DMSO), with the function-blocking anti-α v β 5 -integrin ALULA (α v β 5 -i), with the function-blocking anti-α 5 β 1 -integrin mAB16 (α 5 β 1 -i), with combinations of ALULA + mAb16 (β 5 -i + α 5 -i), with the function-stabilizing anti-active α 5 β 1 -integrin antibody SNAKA51 (α 5 β 1 -act), or with corresponding isotype controls (IgG). ( A ) Representative monochromatic images of immunofluorescently labeled αSMA and corresponding actin stress fibers (F-actin). Checkmarks indicate conditions that induce a myofibroblastic activation phenotype in response to rD-ECM. X marks indicate conditions that did not induce myofibroblastic activation. ( B ) Graph depicting measured levels of stress fiber (F-actin) localized αSMA ratios. The results and statistical analysis for this set of experiments are summarized in . Note that, while TGFβ inhibition rendered matrices produced by CAFs (D + TGFβi) nonfunctional, naïve renal fibroblasts are effectively activated by rD-ECM in a TGFβ-independent manner (TGFβ-i) that is apparently maintained by the same integrin crosstalk seen in the human PDAC model. DOI: http://dx.doi.org/10.7554/eLife.20600.043

Article Snippet: Membranes were then incubated in the one of the following primary antibodies overnight at 4°C: rabbit polyclonal anti-α v -integrin (AB_2631308) (1:5000) (Mybiosource, San Diego, CA), rabbit monoclonal anti-α 5 -integrin (AB_2631309) (1:1000) (Abcam, Cambridge, MA), rabbit polyclonal anti-β 5 -integrin (AB_10806204) (1:1000)(Millipore, Billerica, MA) or rabbit polyclonal anti-β 3 -integrin (1:1000) (AB_91119) (Millipore, Billerica, MA).

Techniques: Cell Culture, Derivative Assay, Produced, Control, Blocking Assay, Labeling, Activation Assay, Inhibition

Journal: eLife

Article Title: Matrix-regulated integrin α v β 5 maintains α 5 β 1 -dependent desmoplastic traits prognostic of neoplastic recurrence

doi: 10.7554/eLife.20600

Figure Lengend Snippet: Naïve r-fibroblasts were re-plated onto rD-ECMs and challenged with either control conditions (DMSO + IgG), small molecule FAK inhibitor PF573,228 (FAK-i) alone or FAK-i in combination with α 5 β 1 -integrin inhibitor (FAK-i + α 5 -i, mAb16 ), and the activation of fibroblasts was tested. ( A ) Representative monochromatic images of immunofluorescently labeled αSMA and actin stress fibers (F-actin). Colored asterisks in ( A ) represent areas that are magnified in the corresponding panels to the right. ( B ) Quantification of αSMA at actin stress fibers (F-actin) from ( A ) normalized to DMSO + IgG control (one arbitrary unit; a.u). (IgG/DMSO vs. FAK-i: ****p=<0.0001, FAK-i vs. FAK-i + α 5 β 1 -i: ***p=0.0051). DOI: http://dx.doi.org/10.7554/eLife.20600.044

Article Snippet: Membranes were then incubated in the one of the following primary antibodies overnight at 4°C: rabbit polyclonal anti-α v -integrin (AB_2631308) (1:5000) (Mybiosource, San Diego, CA), rabbit monoclonal anti-α 5 -integrin (AB_2631309) (1:1000) (Abcam, Cambridge, MA), rabbit polyclonal anti-β 5 -integrin (AB_10806204) (1:1000)(Millipore, Billerica, MA) or rabbit polyclonal anti-β 3 -integrin (1:1000) (AB_91119) (Millipore, Billerica, MA).

Techniques: Control, Activation Assay, Labeling

Journal: eLife

Article Title: Matrix-regulated integrin α v β 5 maintains α 5 β 1 -dependent desmoplastic traits prognostic of neoplastic recurrence

doi: 10.7554/eLife.20600

Figure Lengend Snippet: ( A ) Indirect immunofluorescent and spinning disc confocal generated images of 3D-adhesions, identified using mAb11 , formed by naïve fibroblastic cells cultured within rN-ECM or rD-ECM in the absence (cnt.) or in the presence of ALULA for α v β 5 -integrin inhibition (α v β 5 -i) or of SNAKA51 to stabilize α 5 β 1 -integrin activity (α 5 β 1 act) or of IgG as control. Images were processed using the computer-selected internally threshold objects (ITOs) function of the MetaMorph 7.8.0.0 software. (B) Quantification of the length of ITO-generated objects from ( A ) (**p=0.0492, *p=0.0928). Note the significant differences in 3D-adhesion length observed between rN-ECM and rD-ECM as well as between IgG and SNAKA51 treatments. DOI: http://dx.doi.org/10.7554/eLife.20600.046

Article Snippet: Membranes were then incubated in the one of the following primary antibodies overnight at 4°C: rabbit polyclonal anti-α v -integrin (AB_2631308) (1:5000) (Mybiosource, San Diego, CA), rabbit monoclonal anti-α 5 -integrin (AB_2631309) (1:1000) (Abcam, Cambridge, MA), rabbit polyclonal anti-β 5 -integrin (AB_10806204) (1:1000)(Millipore, Billerica, MA) or rabbit polyclonal anti-β 3 -integrin (1:1000) (AB_91119) (Millipore, Billerica, MA).

Techniques: Generated, Cell Culture, Inhibition, Activity Assay, Control, Software

Journal: eLife

Article Title: Matrix-regulated integrin α v β 5 maintains α 5 β 1 -dependent desmoplastic traits prognostic of neoplastic recurrence

doi: 10.7554/eLife.20600

Figure Lengend Snippet: ( A ) Double-labeled images depicting 3D-adhesion (red in overlay; 3D-adh.) and active α 5 β 1 -integrin (green in overlay; α 5 β 1 -act) in naïve r-fibroblasts cultured overnight in renal normal-ECMs (rN-ECM) or RCC-associated CAF-derived ECMs (rD-ECM). Pseudocolored images at the far right represent semi-quantitative images, maximum reconstructions of active α 5 β 1 -integrin levels (α 5 β 1 -act inten), with a corresponding intensity bar shown on the right. Total active α 5 β 1 -integrin levels were calculated using SMIA-CUKIE, which is publicly available at https://github.com/cukie/SMIA . ( B – D ) Results are summarized in ( B ), using median levels on rD-ECM for normalization (one arbitrary unit; a.u.) (***p=0.0001), in ( C ) using integrated levels of active α 5 β 1 -integrin localized at 3D-adhesions, and in ( D ), using active α 5 β 1 -integrin integrated intensity levels, measured away from 3D-adhesions, in the absence or presence of α v β 5 -integrin inhibition (****p<0.0001). DOI: http://dx.doi.org/10.7554/eLife.20600.047

Article Snippet: Membranes were then incubated in the one of the following primary antibodies overnight at 4°C: rabbit polyclonal anti-α v -integrin (AB_2631308) (1:5000) (Mybiosource, San Diego, CA), rabbit monoclonal anti-α 5 -integrin (AB_2631309) (1:1000) (Abcam, Cambridge, MA), rabbit polyclonal anti-β 5 -integrin (AB_10806204) (1:1000)(Millipore, Billerica, MA) or rabbit polyclonal anti-β 3 -integrin (1:1000) (AB_91119) (Millipore, Billerica, MA).

Techniques: Labeling, Cell Culture, Derivative Assay, Inhibition

Journal: eLife

Article Title: Matrix-regulated integrin α v β 5 maintains α 5 β 1 -dependent desmoplastic traits prognostic of neoplastic recurrence

doi: 10.7554/eLife.20600

Figure Lengend Snippet: ( A ) SMI approach image outputs of in vitro 3D cultures of naïve fibroblastic stellate cells (normal) and desmoplastic CAFs (tumor associated) during ECM production. Leftmost panels demonstrate the positive staining of vimentin (magenta), and the lack of cytokeratin (cyan), indicating the purity of the fibroblasts isolated; nuclei are marked in yellow. White masks (SMI approach; SMIA) in the next panel represent vimentin-positive/cytokeratin-negative (in this case vimentin-positive only as there is no cytokeratin present) areas as recognized by the software following threshold values provided by the user. Next panels represent the assorted markers localized at pixels corresponding to SMI-selected masks and conforming to: active α 5 β 1 -integrin (S-α 5 β 1 act.; in green), 3D-adhesions (S-3D-adh.; in red), pFAK-Y 397 (S-pFAK; in orange), and pSMAD2/3 (S-pSMAD; in blue). ( B ) Graphs summarizing SMIA-CUKIE-generated data outputs representing median intensity levels of active α 5 β 1 -integrin (green bullets), pFAK-Y 397 (orange bullets) and pSMAD2/3 (blue bullets) from data conditions as in ( A ) (***p=0.0002). ( C ) Graphs summarizing SMIA-CUKIE-generated data from marker intersections indicating mean intensity levels of α 5 β 1 -integrin activity localized away from 3D-adhesions (green bullets) (***p=0.0008), pFAK-Y 397 at 3D-adhesions (orange bullets) (***p=0.0002), and nuclear pSMAD 2/3 (blue bullets) (***p=0.0002). DOI: http://dx.doi.org/10.7554/eLife.20600.049

Article Snippet: Membranes were then incubated in the one of the following primary antibodies overnight at 4°C: rabbit polyclonal anti-α v -integrin (AB_2631308) (1:5000) (Mybiosource, San Diego, CA), rabbit monoclonal anti-α 5 -integrin (AB_2631309) (1:1000) (Abcam, Cambridge, MA), rabbit polyclonal anti-β 5 -integrin (AB_10806204) (1:1000)(Millipore, Billerica, MA) or rabbit polyclonal anti-β 3 -integrin (1:1000) (AB_91119) (Millipore, Billerica, MA).

Techniques: In Vitro, Staining, Isolation, Software, Generated, Marker, Activity Assay

Journal: eLife

Article Title: Matrix-regulated integrin α v β 5 maintains α 5 β 1 -dependent desmoplastic traits prognostic of neoplastic recurrence

doi: 10.7554/eLife.20600

Figure Lengend Snippet: Samples correspond to normal fibroblastic stellate cells ( A ) and CAFs ( B ): depicted are spinning disc confocal-acquired images of indirect immunofluorescence of the assorted stromal markers. Images are shown as overlays to indicate the distribution of active α 5 β 1 -integrin (α 5 β 1 act.; green) with regards to 3D-adhesion locations (3D-adh.; red), pFAK-Y 397 (pFAK; orange), pSMAD2/3 (pSMAD; blue) and nucleus (yellow). Inserts correspond to monochromatic images of the corresponding marker. DOI: http://dx.doi.org/10.7554/eLife.20600.050

Article Snippet: Membranes were then incubated in the one of the following primary antibodies overnight at 4°C: rabbit polyclonal anti-α v -integrin (AB_2631308) (1:5000) (Mybiosource, San Diego, CA), rabbit monoclonal anti-α 5 -integrin (AB_2631309) (1:1000) (Abcam, Cambridge, MA), rabbit polyclonal anti-β 5 -integrin (AB_10806204) (1:1000)(Millipore, Billerica, MA) or rabbit polyclonal anti-β 3 -integrin (1:1000) (AB_91119) (Millipore, Billerica, MA).

Techniques: Immunofluorescence, Marker

Journal: eLife

Article Title: Matrix-regulated integrin α v β 5 maintains α 5 β 1 -dependent desmoplastic traits prognostic of neoplastic recurrence

doi: 10.7554/eLife.20600

Figure Lengend Snippet: ( A ) Individual patient SMIA-CUKIE-generated intensity values from PDAC and RCC normal or tumor-associated tissue samples, representing stromal levels of active α 5 β 1 -integrin (green bullets), pFAK-Y 397 (orange bullets) and pSMAD2/3 (blue bullets). ( B ) Stromal active α 5 β 1 -integrin away from 3D-adhesions in pancreatic (left) and renal (right) normal or tumor associated tissues. Significance asterisks represent the following: ( A ) PDAC ***p=0.0013, **p=0.0371; RCC ****p<0.0001, ***p=0.0031; ( B ), **p=0.0371 and ****p<0.0001. DOI: http://dx.doi.org/10.7554/eLife.20600.052

Article Snippet: Membranes were then incubated in the one of the following primary antibodies overnight at 4°C: rabbit polyclonal anti-α v -integrin (AB_2631308) (1:5000) (Mybiosource, San Diego, CA), rabbit monoclonal anti-α 5 -integrin (AB_2631309) (1:1000) (Abcam, Cambridge, MA), rabbit polyclonal anti-β 5 -integrin (AB_10806204) (1:1000)(Millipore, Billerica, MA) or rabbit polyclonal anti-β 3 -integrin (1:1000) (AB_91119) (Millipore, Billerica, MA).

Techniques: Generated

Journal: eLife

Article Title: Matrix-regulated integrin α v β 5 maintains α 5 β 1 -dependent desmoplastic traits prognostic of neoplastic recurrence

doi: 10.7554/eLife.20600

Figure Lengend Snippet: A seven-color simultaneous multi-channel immunofluorescent (SMI; see Materials and methods) approach was implemented to analyze FFPE tissue from the original patient surgical samples from which the fibroblasts used in vitro were harvested. ( A ) Normal tissue panel, representative images of PDAC and RCC FFPE samples. Note that the PDAC images correspond to pathological and matched normal samples from patient #1; fibroblasts harvested from this pair of samples were immortalized and used to generate all human KOs that were presented above. ( B ) Tumor-associated tissue panel, representative images. The first column of images in ( A ) and ( B ) are overlaid images including the three colors used for ‘masked’ locations (see Materials and methods for details); epithelial/tumoral areas are pseudocolored in cyan, stromal vimentin is magenta, and nuclei labeled using draq5 are shown in yellow. The SMIA-CUKIE software ( https://github.com/cukie/SMIA_CUKIE ) was instructed to render an intersection ‘mask’ image (SMIA-mask ‘S’; white) corresponding to pixel areas selected as stroma-positive and epithelial/tumoral-negative to exclude potential mesenchymal to epithelial transduced tumoral locations. Next, to the right of the SMIA mask image, are images of the corresponding markers showing only pixels corresponding to bona fide stromal ‘masks’ depicted in the mask image. Markers shown correspond to: active α 5 β 1 -integrin (S- α 5 β 1 act.; green) and 3D-adhesions (S-3D-adh.; insert red), followed next to the right by pFAK-Y 397 (S-pFAK; orange) and pSMAD2/3 (S-pSMAD; blue). Note that only pixels shown, which corresponded to the selected SMIA-mask (white), were quantitatively analyzed by the SMIA-CUKIE software (see for values). DOI: http://dx.doi.org/10.7554/eLife.20600.051

Article Snippet: Membranes were then incubated in the one of the following primary antibodies overnight at 4°C: rabbit polyclonal anti-α v -integrin (AB_2631308) (1:5000) (Mybiosource, San Diego, CA), rabbit monoclonal anti-α 5 -integrin (AB_2631309) (1:1000) (Abcam, Cambridge, MA), rabbit polyclonal anti-β 5 -integrin (AB_10806204) (1:1000)(Millipore, Billerica, MA) or rabbit polyclonal anti-β 3 -integrin (1:1000) (AB_91119) (Millipore, Billerica, MA).

Techniques: In Vitro, Labeling, Software

Journal: eLife

Article Title: Matrix-regulated integrin α v β 5 maintains α 5 β 1 -dependent desmoplastic traits prognostic of neoplastic recurrence

doi: 10.7554/eLife.20600

Figure Lengend Snippet: CART-generated survival curves depicting recurrence-free survival (RFS) as a function of active stromal α 5 β 1 -integrin levels in PDAC patients. Curves were obtained using ( A ) active α 5 β 1 -integrin median intensity levels and ( B ) percentage area coverage or integrated intensity of stroma populated by active α 5 β 1 -integrin localized at 3D-adhesions that were generated by SMIA-CUKIE, related to RFS. Colored tick-lines crossing Y axes indicate 0.5 survival marks and correspond to X axis locations that mark the median RFS obtained from the curves. P values are shown. All patient data as well as SMIA-CUKIE-generated data corresponding to human cohort constructed TMAs (shown in ) can be found in the online table at the following publically available link : https://www.foxchase.org/sites/fccc/files/assets/cukierman_Franco-Barraza%20SMIA-CUKIE-Dec-2016.xlsx . Note that since our cohorts comprised of samples obtained from surgeries, early neoplastic stages were overrepresented. DOI: http://dx.doi.org/10.7554/eLife.20600.056

Article Snippet: Membranes were then incubated in the one of the following primary antibodies overnight at 4°C: rabbit polyclonal anti-α v -integrin (AB_2631308) (1:5000) (Mybiosource, San Diego, CA), rabbit monoclonal anti-α 5 -integrin (AB_2631309) (1:1000) (Abcam, Cambridge, MA), rabbit polyclonal anti-β 5 -integrin (AB_10806204) (1:1000)(Millipore, Billerica, MA) or rabbit polyclonal anti-β 3 -integrin (1:1000) (AB_91119) (Millipore, Billerica, MA).

Techniques: Generated, Construct

Journal: eLife

Article Title: Matrix-regulated integrin α v β 5 maintains α 5 β 1 -dependent desmoplastic traits prognostic of neoplastic recurrence

doi: 10.7554/eLife.20600

Figure Lengend Snippet: ( A–C ) CART-generated survival curves depicting overall survival (OS) as a function of stromal assorted marker levels in RCC patients. ( A ) Left and middle curves correspond to mean and total intensity levels of stromal pSMAD2/3 and right curves to pSMAD2/3 mean intensity levels particularly localized at stromal nuclei, as a function of OS. ( B ) OS curves as a function of stromal active α 5 β 1 -integrin levels. Top left curves show mean intensity of active α 5 β 1 -integrin related to OS, top middle curves show mean intensity of active α 5 β 1 -integrin at 3D adhesions related to OS, and top right curves show the percentage area coverage of active α 5 β 1 -integrin at 3D adhesions related to OS. Bottom left curves depict median intensity levels of active α 5 β 1 -integrin away from 3D adhesions, while bottom right curves depict percentage area coverage of active α 5 β 1 -integrin away from 3D adhesions. ( C ) Curves depicting OS as a function of stromal pFAK. Top left curves show mean intensity of pFAK related to OS, top middle curves show percentage area coverage of pFAK related to OS, and top right curves show the total intensity pFAK related to OS. Bottom left curves depict median intensity levels of pFAK at 3D adhesions, bottom middle curves depict percentage area coverage of pFAK at 3D adhesions, and bottom right curves depict total intensity of pFAK at 3D adhesions. Colored tick-lines crossing Y axes indicate 0.5 survival marks and correspond to X axis locations that mark the median survival times obtained from the assorted curves. P values are shown. All patient data as well as SMIA-CUKIE-generated data corresponding to human cohort constructed TMAs (shown in ) can be found in the online table at the following publically available link: https://www.foxchase.org/sites/fccc/files/assets/cukierman_Franco-Barraza%20SMIA-CUKIE-Dec-2016.xlsx . Since our cohorts were comprised of samples obtained from surgeries, early neoplastic stages were overrepresented. Note that the above-mentioned links host RCC clinical data that provided the following results, which were obtained prior to CART analyses to assure that in spite of the bias provided by the use of surgical samples, the cohort in question showed significant associations between clinical and outcome variables and OS and Disease-Specific Survival (DSS): associations between pathological stage and OS had HRs of 2.4 (Uni; p=1.11E-10; 95% CI 1.9–3.2) and 3.2 (MVA; p=0.001; 95% CI 1.6–6.2), respectively. Also, Uni analyses of pathological T, N and M showed significant correlation with OS (T —HR = 2.0, p=4.16E-05, 95% CI 1.4–2.8; N — HR = 2.1, p=0.0003, 95% CI 1.4–3.1; and M — HR = 5.4, p=3.79E-10, 95% CI 3.2–9.1). Similarly, Uni and MVA analyses suggested that shorter DSS times are associated with advanced pathological stage (Uni —HR = 3.1, p=7.94E-11 and 95% CI 2.2–4.3; MVA — HR = 3.4, p<0.0001% and 95% CI 1.6–7.3). DOI: http://dx.doi.org/10.7554/eLife.20600.057

Article Snippet: Membranes were then incubated in the one of the following primary antibodies overnight at 4°C: rabbit polyclonal anti-α v -integrin (AB_2631308) (1:5000) (Mybiosource, San Diego, CA), rabbit monoclonal anti-α 5 -integrin (AB_2631309) (1:1000) (Abcam, Cambridge, MA), rabbit polyclonal anti-β 5 -integrin (AB_10806204) (1:1000)(Millipore, Billerica, MA) or rabbit polyclonal anti-β 3 -integrin (1:1000) (AB_91119) (Millipore, Billerica, MA).

Techniques: Generated, Marker, Construct

Journal: eLife

Article Title: Matrix-regulated integrin α v β 5 maintains α 5 β 1 -dependent desmoplastic traits prognostic of neoplastic recurrence

doi: 10.7554/eLife.20600

Figure Lengend Snippet: ( A - C ) CART-generated survival curves depicting disease specific survival (DSS) as a function of stromal assorted marker levels in RCC patients. ( A ) Left and right curves correspond to mean stromal-and stromal-nuclei- localized intensity levels of pSMAD, related to DSS. ( B ) DSS curves as a function of stromal active α 5 β 1 -integrin levels. Top left curves show mean intensity of active α 5 β 1 -integrin related to DSS, top middle curves show stromal percentage active α 5 β 1 -integrin related to DSS, and top right curves show the total stromal intensity levels of active α 5 β 1 -integrin related to DSS. Middle left, middle and right curves depict mean, percentage coverage and total intensity levels of active stromal α 5 β 1 -integrin at 3D adhesions, respectively, each related to DSS. Bottom row from left to right corresponds to mean, median, percentage coverage and total intensity levels of active stromal α 5 β 1 -integrin away from 3D adhesions, each related to DSS. ( C ) Survival curves depicting DSS as a function of stromal pFAK. Top from left to right: curves showing median, percentage coverage and total intensity levels of stromal pFAK related to DSS. Bottom from left to right: curves showing mean, median, percentage coverage and total intensity of stromal pFAK localized at 3D-adhesions related to DSS. Colored tick-lines crossing Y axes indicate 0.5 survival marks and correspond to X axis locations that mark the median survival times obtained from the assorted curves. P values are shown. All patient data as well as SMIA-CUKIE-generated data corresponding to human cohort constructed TMAs (shown in ) can be found in the online table at the following publically available link: https://www.foxchase.org/sites/fccc/files/assets/cukierman_Franco-Barraza%20SMIA-CUKIE-Dec-2016.xlsx . Note that since our cohorts comprised of samples obtained from surgeries, early neoplastic stages were overrepresented. DOI: http://dx.doi.org/10.7554/eLife.20600.058

Article Snippet: Membranes were then incubated in the one of the following primary antibodies overnight at 4°C: rabbit polyclonal anti-α v -integrin (AB_2631308) (1:5000) (Mybiosource, San Diego, CA), rabbit monoclonal anti-α 5 -integrin (AB_2631309) (1:1000) (Abcam, Cambridge, MA), rabbit polyclonal anti-β 5 -integrin (AB_10806204) (1:1000)(Millipore, Billerica, MA) or rabbit polyclonal anti-β 3 -integrin (1:1000) (AB_91119) (Millipore, Billerica, MA).

Techniques: Generated, Marker, Construct

Journal: eLife

Article Title: Matrix-regulated integrin α v β 5 maintains α 5 β 1 -dependent desmoplastic traits prognostic of neoplastic recurrence

doi: 10.7554/eLife.20600

Figure Lengend Snippet: Essentially, naïve fibroblasts grown in N-ECMs do not trigger a surplus in active α V β 5 and α 5 β 1 -integrin conformations. Consequently, the non-pathological N-ECM spares regulation of α V β 5 -integrin activity and assures physiological accumulation of low active α 5 β 1 -integrin levels at the PM, rendering fibroblasts inactive. D-ECMs induce an excess of active α 5 β 1 -integrin and regulate α V β 5 -integrin-mediated relocation of active α 5 β 1 -integrin from the PM to intracellular (e.g., late endosomal) pools, resulting in myofibroblastic activation. Last, this D-ECM activity is dependent on the activity of α V β 5 -integrin, as inhibition of α V β 5 -integrin maintains active α 5 β 1 -integrin at the PM (similarly to N-ECM regulation of active α 5 β 1 -integrin locations), which averts D-ECM-induced myofibroblastic activation. The summarized data provide a possible explanation for the early model presented in , which called for a D-ECM control of α v β 5 regulation of active α 5 β 1 and suggests that the alluded to regulation constitutes a re-localization of active α 5 β 1 to intracellular endosomal compartments, allowing D-ECM-induced naïve-to-myofibroblastic activation. DOI: http://dx.doi.org/10.7554/eLife.20600.059

Article Snippet: Membranes were then incubated in the one of the following primary antibodies overnight at 4°C: rabbit polyclonal anti-α v -integrin (AB_2631308) (1:5000) (Mybiosource, San Diego, CA), rabbit monoclonal anti-α 5 -integrin (AB_2631309) (1:1000) (Abcam, Cambridge, MA), rabbit polyclonal anti-β 5 -integrin (AB_10806204) (1:1000)(Millipore, Billerica, MA) or rabbit polyclonal anti-β 3 -integrin (1:1000) (AB_91119) (Millipore, Billerica, MA).

Techniques: Activity Assay, Activation Assay, Inhibition, Control

Journal: eLife

Article Title: Matrix-regulated integrin α v β 5 maintains α 5 β 1 -dependent desmoplastic traits prognostic of neoplastic recurrence

doi: 10.7554/eLife.20600

Figure Lengend Snippet:

Article Snippet: Membranes were then incubated in the one of the following primary antibodies overnight at 4°C: rabbit polyclonal anti-α v -integrin (AB_2631308) (1:5000) (Mybiosource, San Diego, CA), rabbit monoclonal anti-α 5 -integrin (AB_2631309) (1:1000) (Abcam, Cambridge, MA), rabbit polyclonal anti-β 5 -integrin (AB_10806204) (1:1000)(Millipore, Billerica, MA) or rabbit polyclonal anti-β 3 -integrin (1:1000) (AB_91119) (Millipore, Billerica, MA).

Techniques:

Journal: Cell Reports

Article Title: PRRT2 modulates presynaptic Ca 2+ influx by interacting with P/Q-type channels

doi: 10.1016/j.celrep.2021.109248

Figure Lengend Snippet:

Article Snippet: Anti - Ca v 2.1 alpha-1A Rabbit Polyclonal , Synaptic Systems , Cat. #152203; RRID: AB_2619841.

Techniques: Recombinant, Saline, Protease Inhibitor, BIA-KA, Bradford Assay, Magnetic Beads, Cell Culture, Software