Interaction of kindlin-2 with integrin β3 promotes outside-in signaling responses by the αVβ3 vitronectin receptor by Zhongji Liao, Hisashi Kato, Manjula Pandey, Joseph M. Cantor, Ararat J. Ablooglu, Mark H. Ginsberg, and Sanford J. Shattil Blood Volume 125(12):1995-2004 March 19, 2015 ©2015 by American Society of Hematology

Effects of β3 cytoplasmic tail mutations on β3/protein interactions. Effects of β3 cytoplasmic tail mutations on β3/protein interactions. (A) Amino acid sequences of the β3+/+, β3ΔRGT, and β3/β1(EGK) cytoplasmic tails used in these studies. Pull-down of c-Src, kindlin-2, or talin from (B) immortalized mouse lung endothelial cell lysate (as a convenient source of talin and kindlin-2) or (C) human platelet lysate (as a convenient source of talin and c-Src) by recombinant β3+/+ or mutant β3 cytoplasmic domain model proteins. The αIIb cytoplasmic domain was used as a negative control. Bound talin, kindlin-2, and c-Src were detected by immunoblotting. Loading of pull-down beads with recombinant cytoplasmic domains was monitored by staining with Coomassie brilliant blue. (Left panel) Representative images of pull-downs; (right panel) quantification of binding of kindlin-2 (B) or c-Src (C). Binding of kindlin-2 or c-Src to αIIb or mutant β3 tails was expressed relative to their binding to the β3+/+ tail, which was arbitrarily taken as 1.0. Data represent means ± standard error of the mean (SEM) of 3 independent experiments. **P < .01; *P < .05; NS, not significant (paired Student t test). Zhongji Liao et al. Blood 2015;125:1995-2004 ©2015 by American Society of Hematology

Immortalized β3ΔRGT ECs exhibit reduced migration and angiogenic sprouting. Immortalized β3ΔRGT ECs exhibit reduced migration and angiogenic sprouting. (A-B) Cell migration across vitronectin-coated Transwells. (C-D) Representative images of fibrin gel bead assays to assess EC sprouting from day 1 to day 3; bead diameter, 60-87 μm. (E-F) Quantification of EC sprouting in the 3-dimensional fibrin gel bead assay. Data represent means ± SEM of 4 experiments. *P < .05; NS, not significant (paired Student t test); OD, optical density. Zhongji Liao et al. Blood 2015;125:1995-2004 ©2015 by American Society of Hematology

Enforced interaction of αVβ3ΔRGT with kindlin-2 rescues angiogenic sprouting by ECs. (A) Schematic of the inducible heterodimerizer system used here. Enforced interaction of αVβ3ΔRGT with kindlin-2 rescues angiogenic sprouting by ECs. (A) Schematic of the inducible heterodimerizer system used here. (Top panel) The 2 fusion proteins employed: human αV-dimerizer C and dimerizer A-FLAG-human kindlin-2. β3+/+ or β3ΔRGT immortalized lung endothelial cells were transduced with lentiviruses encoding both of these fusion proteins and an shRNA specific for mouse αV. In theory, after addition of a cell-permeable heterodimerizer (AP21967), human αV-dimerizer C should associate with dimerizer A-human kindlin-2, thereby enforcing interaction between αVβ3 and kindlin-2. (B) Cytodex beads coated with either β3+/+ or β3ΔRGT endothelial cells expressing the dimerizer system described in panel A were incubated overnight at 37°C with 250 nM AP21967 (AP) or vehicle control. Then sprout formation by the cells was evaluated using the 3-dimensional fibrin gel bead assay. Data represent means ± SEM of 4 independent experiments; *P < .05; NS, not significant (paired Student t test). Zhongji Liao et al. Blood 2015;125:1995-2004 ©2015 by American Society of Hematology

β3 interaction with kindlin-2, but not with c-Src, promotes angiogenic sprouting by ECs. (A) Western blots of kindlin-2 and c-Src in β3+/+ immortalized lung endothelial cells transduced with lentivirus encoding kindlin-2 shRNA (shRNA1 and shRNA2), a c-Src s... β3 interaction with kindlin-2, but not with c-Src, promotes angiogenic sprouting by ECs. (A) Western blots of kindlin-2 and c-Src in β3+/+ immortalized lung endothelial cells transduced with lentivirus encoding kindlin-2 shRNA (shRNA1 and shRNA2), a c-Src shRNA, or a control (Ctrl) shRNA. β-actin was monitored as a loading control. (B) Quantification of sprouts by the cells transduced with lentiviruses encoding the kindlin-2 (upper panel), c-Src (lower panel), or Ctrl shRNAs. (C-E) Cells were transduced with kindlin-2 shRNA2 and then transiently transfected with shRNA-resistant complementary DNAs encoding either FLAG-human kindlin-2 (FLAG-hkindlin-2 WT), FLAG-hkindlin-2 QW/AA mutant, or nothing (pcDNA3.1 empty vector). (C) Western blots of human kindlin-2 detected with an anti-FLAG antibody. (D) Flow cytometry histograms depicting intracellular expression of shRNA-resistant kindlin-2 using anti-FLAG antibody. (E) Kindlin-2 rescue of endothelial cell sprout formation in the 3-dimensional fibrin gel bead assay. Note that transfection by electroporation decreased cell vitality so that the number of the sprouts was lower compared with parental immortalized lung endothelial cells. Quantification of EC sprouting is depicted as means ± SEM of 5 experiments. *P < .05; NS, not significant (paired Student t test). Zhongji Liao et al. Blood 2015;125:1995-2004 ©2015 by American Society of Hematology

Deletion of RGT from the β3 cytoplasmic tail does not impair activation of endothelial cell αVβ3. Deletion of RGT from the β3 cytoplasmic tail does not impair activation of endothelial cell αVβ3. (A) Immortalized lung endothelial cells were incubated for 30 minutes at room temperature with WOW-1 Fab in the presence or absence of 200 nM PDBu (to activate protein kinase C) or with 5 mM EDTA (to inhibit specific WOW-1 Fab binding). WOW-1 Fab binding was measured by flow cytometry as described in “Methods.” Data represent specific WOW-1 Fab binding, defined as binding inhibited by EDTA, and represent means ± SEM of 3 independent experiments. **P < .01; *P < .05; NS, not significant (paired Student t test). (B) Localization of activated αVβ3 at lamellipodial edges. Cells were plated onto coverslips coated with fibrinogen and allowed to spread for 1 hour in the presence or absence of 200 nM PDBu as indicated. Cells were fixed, permeabilized, and stained with WOW-1 Fab, with antibody to total β3, and with phalloidin for F-actin as described in “Methods.” Bar represent 30 μm. Zhongji Liao et al. Blood 2015;125:1995-2004 ©2015 by American Society of Hematology

Postnatal retinal angiogenesis is delayed in β3ΔRGT mice, but not in β3/β1(EGK) mice. Postnatal retinal angiogenesis is delayed in β3ΔRGT mice, but not in β3/β1(EGK) mice. Retinas on postnatal day 3 (P3) from β3+/+ and the indicated β3 mutant littermates were stained with anti-CD31 (to mark blood vessels) (A-D) or with fluorescein isothiocyanate–isolectin B4 (for vessels) and anti-Ki67 (for cell proliferation) (E-J) (supplemental Figure 5), and cells were imaged as described in “Methods.” (A,C) Representative images of whole mount retinas with ×10 oil-dipping objective. Bar represents 500 μm. (B,D) Quantification of retinal vessels based on radial length from optic nerve to the periphery. (E,G) Quantification of endothelial tip cells at the retinal vascular front. (F,H) Quantification of endothelial cell filopodial extensions per 25-μm vessel length. (I,J) Quantification of Ki67-positive endothelial cells at the retinal vascular front. (B,D) Data from 12-14 retinas; (E,F,I), data from 8 retinas and 4 mice; (G,H,J) data from 4 retinas and 2 mice. Data are means ± SEM. *P < .05; **P < .01; NS, not significant (unpaired Student t test). Zhongji Liao et al. Blood 2015;125:1995-2004 ©2015 by American Society of Hematology

Tumor growth and tumor vessel density are reduced in β3ΔRGT mice, but not in β3/β1(EGK) mice. β3+/+, β3+/−, and β3 mutant mice were inoculated subcutaneously with 300 μL of 2 × 105 B16F1 melanoma cells. Tumor growth and tumor vessel density are reduced in β3ΔRGT mice, but not in β3/β1(EGK) mice. β3+/+, β3+/−, and β3 mutant mice were inoculated subcutaneously with 300 μL of 2 × 105 B16F1 melanoma cells. (A,C) Quantification of tumor weights 21 days after inoculation. (B,D) Quantification of CD31-positive vessels per field in tumor sections as described in “Methods” (also see supplemental Figure 5C). (E) β3+/+ or β3ΔRGT mice were transplanted with the indicated bone marrow cells. There were 9-10 transplant recipients/group. Four to 6 weeks later, mice were inoculated with tumor cells and tumor weights were quantified 21 days after inoculation. Data represent means ± SEM. *P < .05; **P < .01; NS, not significant (unpaired Student t test). Zhongji Liao et al. Blood 2015;125:1995-2004 ©2015 by American Society of Hematology