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WISP-1 protein promoted Akt phosphorylation via <t>integrin</t> β1/FAK/ILK in human cardiac fibroblasts (HCFs). HCFs were cultured in supplemented fibroblast growth medium for 24 h and then starved in serum-free medium (SFM) for 48 h. The medium was replaced with fresh SFM in the presence or absence of recombinant human WISP-1 protein (500 ng/mL) for 30 min before cell lysis. Cell lysate samples were analysed by Western blotting using phosphorylated Akt (p-Akt) (Ser473) and total Akt (t-Akt) antibodies. ( A ) Representative Western blots of p-Akt (Ser473) and t-Akt protein expression. The ratio of p-Akt (Ser473) to t-Akt was calculated and expressed as the relative fold change to the control. Data shown as mean ± SEM (n = 9). Statistical analysis was performed using Mann–Whitney U test. * indicates p < 0.05. ( B ) Representative Western blots of p-Akt (Ser473) and t-Akt protein expression. The ratio of p-Akt (Ser473) to t-Akt was calculated and expressed as the relative fold change to WISP-1 + mouse non-immune IgG 1 control (mIgG) group. HCFs were pre-incubated with integrin β1-blocking antibodies (mouse IgG 1 clone) (β1 mAb, 10 μg/mL), integrin <t>αVβ5-blocking</t> antibodies (mouse IgG 1 clone) (αVβ5 mAb, 10 μg/mL), and mIgG control antibodies (10 μg/mL), respectively, for 30 min prior to WISP-1 protein treatment. Data shown as mean ± SEM (n = 5). Statistical analysis was performed using Kruskal–Wallis H test. * indicates p < 0.05. ( C ) Representative Western blots of p-Akt (Ser473) and t-Akt protein expression. The ratio of p-Akt (Ser473) to t-Akt was calculated and expressed as the relative fold change to WISP-1 group. HCFs were pre-incubated with defactinib (5 μM) or CPD22 (2.5 μM) for 30 min prior to WISP-1 protein treatment. Data shown as mean ± SEM (n = 4). Statistical analysis was performed using Mann–Whitney U test. * indicates p < 0.05. Approximate molecular weights in kDa are indicated adjacent to representative immunoblots.

Integrin αvβ5 Mab, supplied by Bio-Techne corporation, used in various techniques. Bioz Stars score: 99/100, based on 45 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "WISP-1 Regulates Cardiac Fibrosis by Promoting Cardiac Fibroblasts’ Activation and Collagen Processing"

Article Title: WISP-1 Regulates Cardiac Fibrosis by Promoting Cardiac Fibroblasts’ Activation and Collagen Processing

Journal: Cells

doi: 10.3390/cells13110989

WISP-1 protein promoted Akt phosphorylation via integrin β1/FAK/ILK in human cardiac fibroblasts (HCFs). HCFs were cultured in supplemented fibroblast growth medium for 24 h and then starved in serum-free medium (SFM) for 48 h. The medium was replaced with fresh SFM in the presence or absence of recombinant human WISP-1 protein (500 ng/mL) for 30 min before cell lysis. Cell lysate samples were analysed by Western blotting using phosphorylated Akt (p-Akt) (Ser473) and total Akt (t-Akt) antibodies. ( A ) Representative Western blots of p-Akt (Ser473) and t-Akt protein expression. The ratio of p-Akt (Ser473) to t-Akt was calculated and expressed as the relative fold change to the control. Data shown as mean ± SEM (n = 9). Statistical analysis was performed using Mann–Whitney U test. * indicates p < 0.05. ( B ) Representative Western blots of p-Akt (Ser473) and t-Akt protein expression. The ratio of p-Akt (Ser473) to t-Akt was calculated and expressed as the relative fold change to WISP-1 + mouse non-immune IgG 1 control (mIgG) group. HCFs were pre-incubated with integrin β1-blocking antibodies (mouse IgG 1 clone) (β1 mAb, 10 μg/mL), integrin αVβ5-blocking antibodies (mouse IgG 1 clone) (αVβ5 mAb, 10 μg/mL), and mIgG control antibodies (10 μg/mL), respectively, for 30 min prior to WISP-1 protein treatment. Data shown as mean ± SEM (n = 5). Statistical analysis was performed using Kruskal–Wallis H test. * indicates p < 0.05. ( C ) Representative Western blots of p-Akt (Ser473) and t-Akt protein expression. The ratio of p-Akt (Ser473) to t-Akt was calculated and expressed as the relative fold change to WISP-1 group. HCFs were pre-incubated with defactinib (5 μM) or CPD22 (2.5 μM) for 30 min prior to WISP-1 protein treatment. Data shown as mean ± SEM (n = 4). Statistical analysis was performed using Mann–Whitney U test. * indicates p < 0.05. Approximate molecular weights in kDa are indicated adjacent to representative immunoblots.

Figure Legend Snippet: WISP-1 protein promoted Akt phosphorylation via integrin β1/FAK/ILK in human cardiac fibroblasts (HCFs). HCFs were cultured in supplemented fibroblast growth medium for 24 h and then starved in serum-free medium (SFM) for 48 h. The medium was replaced with fresh SFM in the presence or absence of recombinant human WISP-1 protein (500 ng/mL) for 30 min before cell lysis. Cell lysate samples were analysed by Western blotting using phosphorylated Akt (p-Akt) (Ser473) and total Akt (t-Akt) antibodies. ( A ) Representative Western blots of p-Akt (Ser473) and t-Akt protein expression. The ratio of p-Akt (Ser473) to t-Akt was calculated and expressed as the relative fold change to the control. Data shown as mean ± SEM (n = 9). Statistical analysis was performed using Mann–Whitney U test. * indicates p < 0.05. ( B ) Representative Western blots of p-Akt (Ser473) and t-Akt protein expression. The ratio of p-Akt (Ser473) to t-Akt was calculated and expressed as the relative fold change to WISP-1 + mouse non-immune IgG 1 control (mIgG) group. HCFs were pre-incubated with integrin β1-blocking antibodies (mouse IgG 1 clone) (β1 mAb, 10 μg/mL), integrin αVβ5-blocking antibodies (mouse IgG 1 clone) (αVβ5 mAb, 10 μg/mL), and mIgG control antibodies (10 μg/mL), respectively, for 30 min prior to WISP-1 protein treatment. Data shown as mean ± SEM (n = 5). Statistical analysis was performed using Kruskal–Wallis H test. * indicates p < 0.05. ( C ) Representative Western blots of p-Akt (Ser473) and t-Akt protein expression. The ratio of p-Akt (Ser473) to t-Akt was calculated and expressed as the relative fold change to WISP-1 group. HCFs were pre-incubated with defactinib (5 μM) or CPD22 (2.5 μM) for 30 min prior to WISP-1 protein treatment. Data shown as mean ± SEM (n = 4). Statistical analysis was performed using Mann–Whitney U test. * indicates p < 0.05. Approximate molecular weights in kDa are indicated adjacent to representative immunoblots.

Techniques Used: Cell Culture, Recombinant, Lysis, Western Blot, Expressing, MANN-WHITNEY, Incubation, Blocking Assay

A schematic summary of the findings of this study. WISP-1 promotes cardiac fibroblasts’ phenotypic switch from quiescent fibroblasts to myofibroblasts (activated fibroblasts), promoting collagen processing and accumulation. WISP-1 activates Akt signalling via integrin β1/FAK/ILK in cardiac fibroblasts. Deletion of WISP-1 attenuates angiotensin II (AngII)-induced cardiac fibrotic remodelling in vivo. Figure key is illustrated on the top left-hand side of the figure. Purple ↓ denotes promotion; black ↑ denotes increase; ┤ denotes inhibition.

Figure Legend Snippet: A schematic summary of the findings of this study. WISP-1 promotes cardiac fibroblasts’ phenotypic switch from quiescent fibroblasts to myofibroblasts (activated fibroblasts), promoting collagen processing and accumulation. WISP-1 activates Akt signalling via integrin β1/FAK/ILK in cardiac fibroblasts. Deletion of WISP-1 attenuates angiotensin II (AngII)-induced cardiac fibrotic remodelling in vivo. Figure key is illustrated on the top left-hand side of the figure. Purple ↓ denotes promotion; black ↑ denotes increase; ┤ denotes inhibition.

Techniques Used: In Vivo, Inhibition

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Journal: Cells

Article Title: WISP-1 Regulates Cardiac Fibrosis by Promoting Cardiac Fibroblasts’ Activation and Collagen Processing

doi: 10.3390/cells13110989

Figure Lengend Snippet: WISP-1 protein promoted Akt phosphorylation via integrin β1/FAK/ILK in human cardiac fibroblasts (HCFs). HCFs were cultured in supplemented fibroblast growth medium for 24 h and then starved in serum-free medium (SFM) for 48 h. The medium was replaced with fresh SFM in the presence or absence of recombinant human WISP-1 protein (500 ng/mL) for 30 min before cell lysis. Cell lysate samples were analysed by Western blotting using phosphorylated Akt (p-Akt) (Ser473) and total Akt (t-Akt) antibodies. ( A ) Representative Western blots of p-Akt (Ser473) and t-Akt protein expression. The ratio of p-Akt (Ser473) to t-Akt was calculated and expressed as the relative fold change to the control. Data shown as mean ± SEM (n = 9). Statistical analysis was performed using Mann–Whitney U test. * indicates p < 0.05. ( B ) Representative Western blots of p-Akt (Ser473) and t-Akt protein expression. The ratio of p-Akt (Ser473) to t-Akt was calculated and expressed as the relative fold change to WISP-1 + mouse non-immune IgG 1 control (mIgG) group. HCFs were pre-incubated with integrin β1-blocking antibodies (mouse IgG 1 clone) (β1 mAb, 10 μg/mL), integrin αVβ5-blocking antibodies (mouse IgG 1 clone) (αVβ5 mAb, 10 μg/mL), and mIgG control antibodies (10 μg/mL), respectively, for 30 min prior to WISP-1 protein treatment. Data shown as mean ± SEM (n = 5). Statistical analysis was performed using Kruskal–Wallis H test. * indicates p < 0.05. ( C ) Representative Western blots of p-Akt (Ser473) and t-Akt protein expression. The ratio of p-Akt (Ser473) to t-Akt was calculated and expressed as the relative fold change to WISP-1 group. HCFs were pre-incubated with defactinib (5 μM) or CPD22 (2.5 μM) for 30 min prior to WISP-1 protein treatment. Data shown as mean ± SEM (n = 4). Statistical analysis was performed using Mann–Whitney U test. * indicates p < 0.05. Approximate molecular weights in kDa are indicated adjacent to representative immunoblots.

Article Snippet: In some experiments, HCFs were pre-incubated with a mouse IgG 1 clone antibody (mAb) (10 μg/mL integrin β1 mAb [Biotechne, MAB177781], 10 μg/mL integrin αVβ5 mAb [Biotechne, MAB2528], or 10 μg/mL mouse IgG 1 control antibody [Biotechne, MAB002]), or an inhibitor (5μM defactinib [Abcam, Cambridge, UK, ab254452], 2.5 μM CPD22 [Merck, Darmstadt, Germany, 407331], or 25 μM GM6001 [Tocris, Bristol, UK, 2983]) for 30 min prior to WISP-1 protein treatment.

Techniques: Cell Culture, Recombinant, Lysis, Western Blot, Expressing, MANN-WHITNEY, Incubation, Blocking Assay

Journal: Cells

Article Title: WISP-1 Regulates Cardiac Fibrosis by Promoting Cardiac Fibroblasts’ Activation and Collagen Processing

doi: 10.3390/cells13110989

Figure Lengend Snippet: A schematic summary of the findings of this study. WISP-1 promotes cardiac fibroblasts’ phenotypic switch from quiescent fibroblasts to myofibroblasts (activated fibroblasts), promoting collagen processing and accumulation. WISP-1 activates Akt signalling via integrin β1/FAK/ILK in cardiac fibroblasts. Deletion of WISP-1 attenuates angiotensin II (AngII)-induced cardiac fibrotic remodelling in vivo. Figure key is illustrated on the top left-hand side of the figure. Purple ↓ denotes promotion; black ↑ denotes increase; ┤ denotes inhibition.

Techniques: In Vivo, Inhibition

αvβ5-integrin and chondroitin sulfate proteoglycans are implicated in Wnt1-inducible signaling protein 1 (WISP1)-induced production of migration inhibitory factor (MIF) from human lung fibroblasts (HLFs). HLFs were pretreated for 30 minutes with anti-αvβ5-integrin antibody (6 µg/mL) ( A ) or for 2 h with chondroitinase ABC or chondroitinase AC II (3 units/mL) ( B ), followed by stimulation with WISP1 (100 ng/mL) for 48 h, and the levels of MIF in conditioned media were determined by ELISA. Data represent the means ± SD ( n = 3). *Statistically significant increase ( P < 0.05) compared to control. #Statistically significant decrease ( P < 0.05) compared to WISP1-treated cells. IgG represents immunoglobulins from nonimmunized mouse.

Journal: American Journal of Physiology - Cell Physiology

Article Title: WISP1 induces the expression of macrophage migration inhibitory factor in human lung fibroblasts through Src kinases and EGFR-activated signaling pathways

doi: 10.1152/ajpcell.00410.2023

Figure Lengend Snippet: αvβ5-integrin and chondroitin sulfate proteoglycans are implicated in Wnt1-inducible signaling protein 1 (WISP1)-induced production of migration inhibitory factor (MIF) from human lung fibroblasts (HLFs). HLFs were pretreated for 30 minutes with anti-αvβ5-integrin antibody (6 µg/mL) ( A ) or for 2 h with chondroitinase ABC or chondroitinase AC II (3 units/mL) ( B ), followed by stimulation with WISP1 (100 ng/mL) for 48 h, and the levels of MIF in conditioned media were determined by ELISA. Data represent the means ± SD ( n = 3). *Statistically significant increase ( P < 0.05) compared to control. #Statistically significant decrease ( P < 0.05) compared to WISP1-treated cells. IgG represents immunoglobulins from nonimmunized mouse.

Article Snippet: Recombinant human EGF and anti-integrin αvβ5 mouse mAb (no. MAB2528-SP) were purchased from R&D Systems (Bio-Techne Ltd., Abingdon, UK).

Techniques: Migration, Enzyme-linked Immunosorbent Assay, Control

Proposed model of the effects of Wnt1-inducible signaling protein 1 (WISP1) on the expression of migration inhibitory factor (MIF) and its receptors, as well as on the expression of cyclooxygenase (COX)-2, IL-6, and matrix metalloproteinase (MMP)-2, and production of prostaglandin E 2 (PGE 2 ) from human lung fibroblasts (HLFs). The effect of WISP-1 on the expression of MIF in HLFs with the complex interactions between WISP-1, αvβ5, Src kinases, EGF receptor (EGFR), and CD74/CD44 receptors, reveal intricate regulatory networks that could contribute to the expression of proinflamamtory molecules and remodeling enzymes secretion.

Journal: American Journal of Physiology - Cell Physiology

Article Title: WISP1 induces the expression of macrophage migration inhibitory factor in human lung fibroblasts through Src kinases and EGFR-activated signaling pathways

doi: 10.1152/ajpcell.00410.2023

Figure Lengend Snippet: Proposed model of the effects of Wnt1-inducible signaling protein 1 (WISP1) on the expression of migration inhibitory factor (MIF) and its receptors, as well as on the expression of cyclooxygenase (COX)-2, IL-6, and matrix metalloproteinase (MMP)-2, and production of prostaglandin E 2 (PGE 2 ) from human lung fibroblasts (HLFs). The effect of WISP-1 on the expression of MIF in HLFs with the complex interactions between WISP-1, αvβ5, Src kinases, EGF receptor (EGFR), and CD74/CD44 receptors, reveal intricate regulatory networks that could contribute to the expression of proinflamamtory molecules and remodeling enzymes secretion.

Article Snippet: Recombinant human EGF and anti-integrin αvβ5 mouse mAb (no. MAB2528-SP) were purchased from R&D Systems (Bio-Techne Ltd., Abingdon, UK).

Techniques: Expressing, Migration

Blockade of Tat entry in IC-HUVEC by antibodies directed against the α5β1, αvβ3, and/or αvβ5 integrin. In ( A ), activated HUVEC were suspended and then incubated for 2 h with 5 μg/mL of monoclonal antibodies (mAb) directed against CD31 (upper left panel), α5β1 (upper right panel), or αvβ3 (lower left panel), or with anti-α5β1 and anti-αvβ3 mAb, combined together at 5 μg/mL each (lower right panel). In ( B ), IC-HUVEC were suspended and then incubated for 2 h with 5 μg/mL of anti-α6β4 mAb (upper left panel), or with 5 μg/mL of anti-αvβ5 mAb, alone (upper right panel) or combined with anti-α5β1 and anti-αvβ3 mAb (lower panel). For both ( A , B ), IC-HUVEC incubated with Ab dilution buffer (PBS-0.1% BSA) were employed as controls (Nil). After incubation, IC-HUVEC was exposed for 10 min to biologically active Tat (1–1000 ng/mL) or its buffer. Intracellular Tat content was assayed by intracellular staining and flow cytometry as described. Results are expressed as the percentage of positive cells as compared to isotype-stained samples and non-permeabilized cells. Box-plot data obtained from three to four independent experiments and analyzed by the Mann–Whitney test are shown. Dots indicate individual measures. Orange plots refer to IC-HUVEC incubated with the said mAbs; blue plots refer to control IC-HUVEC.

Journal: International Journal of Molecular Sciences

Article Title: HIV-1 Tat Protein Enters Dysfunctional Endothelial Cells via Integrins and Renders Them Permissive to Virus Replication

doi: 10.3390/ijms22010317

Figure Lengend Snippet: Blockade of Tat entry in IC-HUVEC by antibodies directed against the α5β1, αvβ3, and/or αvβ5 integrin. In ( A ), activated HUVEC were suspended and then incubated for 2 h with 5 μg/mL of monoclonal antibodies (mAb) directed against CD31 (upper left panel), α5β1 (upper right panel), or αvβ3 (lower left panel), or with anti-α5β1 and anti-αvβ3 mAb, combined together at 5 μg/mL each (lower right panel). In ( B ), IC-HUVEC were suspended and then incubated for 2 h with 5 μg/mL of anti-α6β4 mAb (upper left panel), or with 5 μg/mL of anti-αvβ5 mAb, alone (upper right panel) or combined with anti-α5β1 and anti-αvβ3 mAb (lower panel). For both ( A , B ), IC-HUVEC incubated with Ab dilution buffer (PBS-0.1% BSA) were employed as controls (Nil). After incubation, IC-HUVEC was exposed for 10 min to biologically active Tat (1–1000 ng/mL) or its buffer. Intracellular Tat content was assayed by intracellular staining and flow cytometry as described. Results are expressed as the percentage of positive cells as compared to isotype-stained samples and non-permeabilized cells. Box-plot data obtained from three to four independent experiments and analyzed by the Mann–Whitney test are shown. Dots indicate individual measures. Orange plots refer to IC-HUVEC incubated with the said mAbs; blue plots refer to control IC-HUVEC.

Article Snippet: MAbs directed against the α5β1, αvβ3, αvβ5, or α6β4 integrins were obtained from Chemicon-Merck-Millipore (Darmstadt, Germany).

Techniques: Incubation, Bioprocessing, Staining, Flow Cytometry, MANN-WHITNEY, Control

Influence of PAK4 transient or stable overexpression on CMAC size and number. (A) Images from control MCF-7 cells or MCF-7 cells transiently expressing HA-PAK4-WT 3 h after replating onto VN, stained for integrin αvβ5 and HA-tag as indicated. Bar, 20 μm. (B) Quantification of CMAC numbers at the cellular periphery. The results are displayed as means ± SEM of the number of peripheral CMACs within 5 μm of the cell border per cell; between three independent experiments. (C and D) Quantification of CMAC sizes at the cellular periphery. The sizes of the CMACs are categorized into small (≤1 μm 2 ), medium (from >1 to ≤2 μm 2 ), and large (>2 μm 2 ) adhesions. Values represent the percentage distribution (C) or number per cell (D) for each group expressed as mean ± SEM between three independent experiments. (E and G) MCF-7 cells stably expressing Flag-BAP or Flag-PAK4-WT were plated onto VN and fixed after 6 h. Cells were stained with anti-αvβ5 (E) or anti-vinculin (G) antibodies and costained with rhodamine-phalloidin. Bar, 25 μm. (F and H) Quantification of the number of CMACs at the cellular periphery as described in Materials and Methods. Graphs show means of CMACs per cell ± 95% confidence intervals; n = 48 (F) and n = 40 (H). In addition to effects by PAK4 on CMACs, we observed fewer actin stress fibers and also an induction of filopodia (arrows) in some WT PAK4-overexpressing cells compared with controls. p values indicated according to unpaired two-tailed t test.

Journal: Molecular Biology of the Cell

Article Title: Integrin-mediated Cell Attachment Induces a PAK4-dependent Feedback Loop Regulating Cell Adhesion through Modified Integrin αvβ5 Clustering and Turnover

doi: 10.1091/mbc.E10-03-0245

Figure Lengend Snippet: Influence of PAK4 transient or stable overexpression on CMAC size and number. (A) Images from control MCF-7 cells or MCF-7 cells transiently expressing HA-PAK4-WT 3 h after replating onto VN, stained for integrin αvβ5 and HA-tag as indicated. Bar, 20 μm. (B) Quantification of CMAC numbers at the cellular periphery. The results are displayed as means ± SEM of the number of peripheral CMACs within 5 μm of the cell border per cell; between three independent experiments. (C and D) Quantification of CMAC sizes at the cellular periphery. The sizes of the CMACs are categorized into small (≤1 μm 2 ), medium (from >1 to ≤2 μm 2 ), and large (>2 μm 2 ) adhesions. Values represent the percentage distribution (C) or number per cell (D) for each group expressed as mean ± SEM between three independent experiments. (E and G) MCF-7 cells stably expressing Flag-BAP or Flag-PAK4-WT were plated onto VN and fixed after 6 h. Cells were stained with anti-αvβ5 (E) or anti-vinculin (G) antibodies and costained with rhodamine-phalloidin. Bar, 25 μm. (F and H) Quantification of the number of CMACs at the cellular periphery as described in Materials and Methods. Graphs show means of CMACs per cell ± 95% confidence intervals; n = 48 (F) and n = 40 (H). In addition to effects by PAK4 on CMACs, we observed fewer actin stress fibers and also an induction of filopodia (arrows) in some WT PAK4-overexpressing cells compared with controls. p values indicated according to unpaired two-tailed t test.

Article Snippet: The anti-HA (Y11) pAb was purchased from Santa Cruz Biotechnology (Santa Cruz, CA); anti-integrin αvβ5 mAb (clone 15F11) from Chemicon International (Temecula, CA); anti-Flag mAb M2 from Sigma (St. Louis, MO); and tubulin-α Ab-2 mAb from Lab Vision (Fremont, CA).

Techniques: Over Expression, Expressing, Staining, Stable Transfection, Two Tailed Test

Quantitative image analysis of CMAC component intensity and colocalization. Three-channel confocal images were acquired of a single MCF7 cell expressing either EGFP (A–F) or EGFP-PAK4 (J–O) and labeled for integrin αvβ5 (A, D, J, and M) and F-actin (B, E, K, and N). Cell boundaries were defined using F-actin labeling (green lines in images D, E, M, and N cropped from yellow regions of images A, B, J, and K). CMACs within these boundaries were then detected and defined using the integrin αvβ5 channel (red outlines in D, E, and M, N; blue lines within individual CMACs indicate CMAC major axes). CMAC center of mass coordinates (X and Y), CMAC area, mean CMAC intensity (αvβ5 and F-actin), and intra-CMAC colocalization (as defined by Pearson's r within each CMAC) of αvβ5 and F-actin were measured. Cropped, merged raw images of integrin αvβ5 (green) and F-actin (red) as well as either EGFP (blue, F) or EGFP-PAK4 (blue, O) show F-actin extending in protrusive structures beyond the existing CMACs, as well as strong colocalization (yellow) between integrin αvβ5 and F-actin in EGFP- but not EGFP-PAK4–expressing cells. Quantitative data (G–I and P–R) derived from cropped regions show each detected CMAC distributed according to its original X,Y coordinates, with dot size quantitatively reflecting original CMAC area. Dot color and associated number indicate mean integrin αvβ5 intensity (G, P), mean F-actin intensity (H and Q), or intra-CMAC colocalization of αvβ5 and F-actin (I and R), per CMAC. Color scales for αvβ5 intensity, F-actin intensity, and αvβ5/F-actin colocalization are shown to the right of P, Q, and R, respectively. These data demonstrate the method of quantitative data extraction and show directly the visual and quantitative evidence for reduced colocalization between F-actin and integrin αvβ5 within CMACs in cells overexpressing EGFP-PAK4 (see where equivalent data for 100s of cells and 1000s of CMACs are summarized). (S) The outcomes of Pearson's r analyses of two-channel colocalization. The distributions of red and green intensity information within Adhesion “X” reflect a strong spatial and intensity correlation between these two channels, resulting in a positive correlation score (a perfect positive correlation = 1; however, much lower values are typically detected in biological images due largely to poor signal to noise ratios). Red and green intensity distributions within Adhesion “Y” are inversed, resulting in a negative correlation (a perfect negative correlation = −1). Red and green intensity distributions appear unrelated in Adhesion “Z”, resulting in an r-value close to zero.

Journal: Molecular Biology of the Cell

Article Title: Integrin-mediated Cell Attachment Induces a PAK4-dependent Feedback Loop Regulating Cell Adhesion through Modified Integrin αvβ5 Clustering and Turnover

doi: 10.1091/mbc.E10-03-0245

Figure Lengend Snippet: Quantitative image analysis of CMAC component intensity and colocalization. Three-channel confocal images were acquired of a single MCF7 cell expressing either EGFP (A–F) or EGFP-PAK4 (J–O) and labeled for integrin αvβ5 (A, D, J, and M) and F-actin (B, E, K, and N). Cell boundaries were defined using F-actin labeling (green lines in images D, E, M, and N cropped from yellow regions of images A, B, J, and K). CMACs within these boundaries were then detected and defined using the integrin αvβ5 channel (red outlines in D, E, and M, N; blue lines within individual CMACs indicate CMAC major axes). CMAC center of mass coordinates (X and Y), CMAC area, mean CMAC intensity (αvβ5 and F-actin), and intra-CMAC colocalization (as defined by Pearson's r within each CMAC) of αvβ5 and F-actin were measured. Cropped, merged raw images of integrin αvβ5 (green) and F-actin (red) as well as either EGFP (blue, F) or EGFP-PAK4 (blue, O) show F-actin extending in protrusive structures beyond the existing CMACs, as well as strong colocalization (yellow) between integrin αvβ5 and F-actin in EGFP- but not EGFP-PAK4–expressing cells. Quantitative data (G–I and P–R) derived from cropped regions show each detected CMAC distributed according to its original X,Y coordinates, with dot size quantitatively reflecting original CMAC area. Dot color and associated number indicate mean integrin αvβ5 intensity (G, P), mean F-actin intensity (H and Q), or intra-CMAC colocalization of αvβ5 and F-actin (I and R), per CMAC. Color scales for αvβ5 intensity, F-actin intensity, and αvβ5/F-actin colocalization are shown to the right of P, Q, and R, respectively. These data demonstrate the method of quantitative data extraction and show directly the visual and quantitative evidence for reduced colocalization between F-actin and integrin αvβ5 within CMACs in cells overexpressing EGFP-PAK4 (see where equivalent data for 100s of cells and 1000s of CMACs are summarized). (S) The outcomes of Pearson's r analyses of two-channel colocalization. The distributions of red and green intensity information within Adhesion “X” reflect a strong spatial and intensity correlation between these two channels, resulting in a positive correlation score (a perfect positive correlation = 1; however, much lower values are typically detected in biological images due largely to poor signal to noise ratios). Red and green intensity distributions within Adhesion “Y” are inversed, resulting in a negative correlation (a perfect negative correlation = −1). Red and green intensity distributions appear unrelated in Adhesion “Z”, resulting in an r-value close to zero.

Techniques: Expressing, Labeling, Derivative Assay

Influence of PAK4 overexpression on CMAC size, number, integrin clustering density, and integrin–actin connectivity. (A) Quantification of CMAC numbers at the cellular periphery from MCF-7 cells transiently expressing EGFP or EGFP-PAK4-WT. Graph shows means of the number of peripheral CMACs per cell ± 95% confidence intervals from 47 (EGFP control cells) and 48 (PAK4-overexpressing cells). (B) Quantification of integrin clustering density in CMACs at the cellular periphery. The mean intensity of endogenous αvβ5 labeling in CMACs <5 μm from the cell border. The results are displayed as means of all CMACs in the designated size classes (μm 2 ) ± 95% confidence intervals. (C) Quantification of αvβ5 versus F-actin colocalization in CMACs <5 μm from the cell border. Calculated and displayed per CMAC using Pearson's r (r) ± 95% confidence intervals. (D) Quantification of F-actin mean intensity (labeled by phalloidin) in CMACs (the area overlaying αvβ5 labeling) <5 μm from the cell border as displayed in G. Panels G, H, and I in are representative images showing the measurement of 4036 CMACs taken from 47 EGFP control cells and 2789 CMACs from 48 EGFP-PAK4-overexpressing cells. All data in are derived from at least three distinct experiments.

Journal: Molecular Biology of the Cell

Article Title: Integrin-mediated Cell Attachment Induces a PAK4-dependent Feedback Loop Regulating Cell Adhesion through Modified Integrin αvβ5 Clustering and Turnover

doi: 10.1091/mbc.E10-03-0245

Figure Lengend Snippet: Influence of PAK4 overexpression on CMAC size, number, integrin clustering density, and integrin–actin connectivity. (A) Quantification of CMAC numbers at the cellular periphery from MCF-7 cells transiently expressing EGFP or EGFP-PAK4-WT. Graph shows means of the number of peripheral CMACs per cell ± 95% confidence intervals from 47 (EGFP control cells) and 48 (PAK4-overexpressing cells). (B) Quantification of integrin clustering density in CMACs at the cellular periphery. The mean intensity of endogenous αvβ5 labeling in CMACs <5 μm from the cell border. The results are displayed as means of all CMACs in the designated size classes (μm 2 ) ± 95% confidence intervals. (C) Quantification of αvβ5 versus F-actin colocalization in CMACs <5 μm from the cell border. Calculated and displayed per CMAC using Pearson's r (r) ± 95% confidence intervals. (D) Quantification of F-actin mean intensity (labeled by phalloidin) in CMACs (the area overlaying αvβ5 labeling) <5 μm from the cell border as displayed in G. Panels G, H, and I in are representative images showing the measurement of 4036 CMACs taken from 47 EGFP control cells and 2789 CMACs from 48 EGFP-PAK4-overexpressing cells. All data in are derived from at least three distinct experiments.

Techniques: Over Expression, Expressing, Labeling, Derivative Assay

Influence of PAK4 knockdown on CMAC size, number, integrin-clustering density, and actin content. (A) MCF-7 cells stably expressing PAK4-shRNA or control shRNA were plated onto VN and fixed 3 h after replating. Cells were stained with an anti-integrin αvβ5 antibody and costained with rhodamine-phalloidin. Bar, 20 μm. (B) Quantification of the number of CMACs <5 μm from the cellular periphery. The results are presented as mean of number of CMACs per cell ± 95% confidence intervals; n = 36 (shRNA control cells) and 40 (PAK4-shRNA cells); p values according to unpaired two-tailed t test. (C) Distribution of CMAC sizes <5 μm from the cellular periphery; fold change in frequency of different CMAC size classes. (D) Quantification of integrin clustering density in CMACs at the cellular periphery. The mean intensity of endogenous αvβ5 labeling in CMACs <5 μm from the cell border was analyzed. The results are displayed as means of all CMACs in the designated size classes (μm 2 ) ± 95% confidence intervals. (E) Quantification of F-actin mean pixel intensity (labeled by phalloidin) in CMACs (the area overlaying αvβ5 labeling) <5 μm from the cell border, displayed as in D. (F) Quantification of αvβ5 versus F-actin colocalization in CMACs <5 μm from the cell border. Calculated and displayed per CMAC using Pearson's r (r) ± 95% confidence intervals. Data for D–F are derived from three independent experiments measuring 4666 CMACs (36 shRNA control cells) and 9235 CMACs (40 PAK4 shRNA cells). All of the experiments in were repeated at least three times with similar results.

Journal: Molecular Biology of the Cell

Article Title: Integrin-mediated Cell Attachment Induces a PAK4-dependent Feedback Loop Regulating Cell Adhesion through Modified Integrin αvβ5 Clustering and Turnover

doi: 10.1091/mbc.E10-03-0245

Figure Lengend Snippet: Influence of PAK4 knockdown on CMAC size, number, integrin-clustering density, and actin content. (A) MCF-7 cells stably expressing PAK4-shRNA or control shRNA were plated onto VN and fixed 3 h after replating. Cells were stained with an anti-integrin αvβ5 antibody and costained with rhodamine-phalloidin. Bar, 20 μm. (B) Quantification of the number of CMACs <5 μm from the cellular periphery. The results are presented as mean of number of CMACs per cell ± 95% confidence intervals; n = 36 (shRNA control cells) and 40 (PAK4-shRNA cells); p values according to unpaired two-tailed t test. (C) Distribution of CMAC sizes <5 μm from the cellular periphery; fold change in frequency of different CMAC size classes. (D) Quantification of integrin clustering density in CMACs at the cellular periphery. The mean intensity of endogenous αvβ5 labeling in CMACs <5 μm from the cell border was analyzed. The results are displayed as means of all CMACs in the designated size classes (μm 2 ) ± 95% confidence intervals. (E) Quantification of F-actin mean pixel intensity (labeled by phalloidin) in CMACs (the area overlaying αvβ5 labeling) <5 μm from the cell border, displayed as in D. (F) Quantification of αvβ5 versus F-actin colocalization in CMACs <5 μm from the cell border. Calculated and displayed per CMAC using Pearson's r (r) ± 95% confidence intervals. Data for D–F are derived from three independent experiments measuring 4666 CMACs (36 shRNA control cells) and 9235 CMACs (40 PAK4 shRNA cells). All of the experiments in were repeated at least three times with similar results.

Techniques: Stable Transfection, Expressing, shRNA, Staining, Two Tailed Test, Labeling, Derivative Assay

PAK4 promotes integrin αvβ5 turnover within CMACs. FRAP analysis of integrin β5-EGFP turnover in focal adhesions of (A) MCF-7 cells coexpressing mRFP and β5-EGFP or (B) mRFP-PAK4 and β5-EGFP. Enlarged images are shown (from boxes in A and B) containing individually bleached adhesions before bleaching, immediately after bleaching, and after 900-s recovery (arrowheads in bottom panels of A and B indicates bleached focal adhesions). Bar, 25 μm. (C) Quantified integrin β5-EGFP recovery in CMACs after bleaching of cells coexpressing mRFP-PAK4 or mRFP control. The mean fluorescence intensity in the bleached region was quantified and expressed as the percentage recovery relative to the mean of three prebleached values (for that region). Background diffuse integrin intensity within the plasma membrane was subtracted from all values and further corrections were applied for nonspecific bleaching. Values represent means ± SEM from three experiments, each with a minimum of five cells per condition, with a total of 100 adhesions analyzed after photobleaching. Statistically discernable difference between mRFP and mRFP-PAK4 recovery curves was assessed at each time point; with p = 0.018 at 30 s after bleaching, and p < 0.001 at all times after 30 s according to a two-tailed unpaired t test. (D) Equivalent analysis of β5-EGFP recovery after bleaching of plasma membrane regions devoid of CMACs in cells coexpressing mRFP-PAK4 or mRFP control. Values represent means from three distinct experiments including eight cells and 13 bleached regions per condition. A two-tailed unpaired t test of the means at all time points reveals a statistically discernable difference p = 0.011, and t test using all samples and all time points indicates p = 1.7 × 10 −9 . Fitted lines represent free diffusion recovery functions as previously described ( Scott et al. , 2006 ). (E and F) Analysis of the first order recovery (excluding the first 120 s after bleaching that include recovery from free diffusion within the plasma membrane) of β5-EGFP in CMACs reveals significantly enhanced percentage of recovery (E) and recovery rate (F) in the presence of mRFP-PAK4. p values were calculated using two-tailed unpaired t tests. Representative FRAP Movies for are presented in the supplementary information.

Journal: Molecular Biology of the Cell

Article Title: Integrin-mediated Cell Attachment Induces a PAK4-dependent Feedback Loop Regulating Cell Adhesion through Modified Integrin αvβ5 Clustering and Turnover

doi: 10.1091/mbc.E10-03-0245

Figure Lengend Snippet: PAK4 promotes integrin αvβ5 turnover within CMACs. FRAP analysis of integrin β5-EGFP turnover in focal adhesions of (A) MCF-7 cells coexpressing mRFP and β5-EGFP or (B) mRFP-PAK4 and β5-EGFP. Enlarged images are shown (from boxes in A and B) containing individually bleached adhesions before bleaching, immediately after bleaching, and after 900-s recovery (arrowheads in bottom panels of A and B indicates bleached focal adhesions). Bar, 25 μm. (C) Quantified integrin β5-EGFP recovery in CMACs after bleaching of cells coexpressing mRFP-PAK4 or mRFP control. The mean fluorescence intensity in the bleached region was quantified and expressed as the percentage recovery relative to the mean of three prebleached values (for that region). Background diffuse integrin intensity within the plasma membrane was subtracted from all values and further corrections were applied for nonspecific bleaching. Values represent means ± SEM from three experiments, each with a minimum of five cells per condition, with a total of 100 adhesions analyzed after photobleaching. Statistically discernable difference between mRFP and mRFP-PAK4 recovery curves was assessed at each time point; with p = 0.018 at 30 s after bleaching, and p < 0.001 at all times after 30 s according to a two-tailed unpaired t test. (D) Equivalent analysis of β5-EGFP recovery after bleaching of plasma membrane regions devoid of CMACs in cells coexpressing mRFP-PAK4 or mRFP control. Values represent means from three distinct experiments including eight cells and 13 bleached regions per condition. A two-tailed unpaired t test of the means at all time points reveals a statistically discernable difference p = 0.011, and t test using all samples and all time points indicates p = 1.7 × 10 −9 . Fitted lines represent free diffusion recovery functions as previously described ( Scott et al. , 2006 ). (E and F) Analysis of the first order recovery (excluding the first 120 s after bleaching that include recovery from free diffusion within the plasma membrane) of β5-EGFP in CMACs reveals significantly enhanced percentage of recovery (E) and recovery rate (F) in the presence of mRFP-PAK4. p values were calculated using two-tailed unpaired t tests. Representative FRAP Movies for are presented in the supplementary information.

Techniques: Fluorescence, Two Tailed Test, Diffusion-based Assay

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