Journal: Analytical Cellular Pathology (Amsterdam)

Article Title: YAP Overexpression in Breast Cancer Cells Promotes Angiogenesis through Activating YAP Signaling in Vascular Endothelial Cells

doi: 10.1155/2022/5942379

Figure Lengend Snippet: Conditioned medium from YAP overexpressed breast cancer cells promoted tube formation, migration, and proliferation of human umbilical vein endothelial cells (HUVECs). (a) MDA-MB-231 cells were transfected with YAP CRISPR/Cas9 KO plasmid (YAP KD) and YAP CRISPR activation plasmids (YAP OE), YAP expression was detected by Western blot. (b) After starved from FBS for 16 hr, human umbilical vein endothelial cells (HUVECs) were treated with conditioned medium obtained from YAP knock down (CM-YAP-) and overexpressed (CM-YAP+) breast cancer cells for 12 hr. The total segment length and total branching length from tube formation assay are quantified with means ± SD from six independent representative fields. ∗∗∗ p < 0.001. (c) Migration of HUVECs with different conditioned medium treatment was conducted by Transwell assay. The results are from three independent experiments. ∗∗ p < 0.01, ∗∗∗ p < 0.001. (d) HUVECs were treated with different conditioned medium. Cell proliferation was tested by plate cloning formation. ∗∗ p < 0.01. The cloning formation rate is quantified with mean ± SD from six independent wells. ∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: Cells were transfected with YAP CRISPR/Cas9 KO plasmid and YAP CRISPR activation plasmids using UltraCruz® Transfection Reagent (Santa Cruz, USA) according to manufacturer's instruction.

Techniques: Migration, Transfection, CRISPR, Plasmid Preparation, Activation Assay, Expressing, Western Blot, Knockdown, Tube Formation Assay, Transwell Assay, Cloning

Journal: Analytical Cellular Pathology (Amsterdam)

Article Title: YAP Overexpression in Breast Cancer Cells Promotes Angiogenesis through Activating YAP Signaling in Vascular Endothelial Cells

doi: 10.1155/2022/5942379

Figure Lengend Snippet: YAP expression in HUVECs involved in mediating CM-YAP+ induced tube formation, migration, and proliferation of HUVECs. (a) HUVECs were transfected with YAP CRISPR/Cas9 KO plasmid (YAP KD), YAP expression was detected by Western blot. (b) After starved from FBS for 16 hr, HUVECs, and YAP knock down (KD) HUVECs were treated with different conditioned medium obtained from breast cancer cells for 12 hr. The total segment length and total branching length are quantified with mean ± SD from six independent representative fields ∗ p < 0.05, ∗∗ p < 0.01. (c) Migration of HUVECs and YAP knock down HUVECs with different conditioned medium treatment was conducted by Transwell assay. The results are from three independent experiments, ∗∗ p < 0.01. (d) HUVECs and YAP KD HUVECs were treated with different conditioned medium. Cell proliferation was tested by plate cloning formation. The cloning formation rate is quantified with mean ± SD from six independent wells. ∗∗ p < 0.01.

Article Snippet: Cells were transfected with YAP CRISPR/Cas9 KO plasmid and YAP CRISPR activation plasmids using UltraCruz® Transfection Reagent (Santa Cruz, USA) according to manufacturer's instruction.

Techniques: Expressing, Migration, Transfection, CRISPR, Plasmid Preparation, Western Blot, Knockdown, Transwell Assay, Cloning

Journal: Molecular and Cellular Biology

Article Title: CCN1–Yes-Associated Protein Feedback Loop Regulates Physiological and Pathological Angiogenesis

doi: 10.1128/MCB.00107-19

Figure Lengend Snippet: EC-specific deletion of CCN1 increased YAP activation and EC proliferation during vessel development. (A) Schematic representation of EC-specific deletion of CCN1 in mice. (B) CCN1 mRNA levels in CCN1+/+ and EC-ΔCCN1 mouse retinas. **, P < 0.001 versus CCN1+/+ mice (n = 4). (C and D) Expression of CCN1, pYAP, and YAP in retinal protein lysates from control CCN1+/+ and EC-ΔCCN1 mice. **, P < 0.001 versus YAP of CCN1+/+ mice (n = 4). (E) IB4-stained retinal flat mounts of CCN1+/+ and EC-ΔCCN1 mice. (F) The outline of blood vessels, the vasculature skeleton, and the branching points are highlighted in yellow, red, and green, respectively. (G and H) Quantitative analysis of vascular areas and junctional density of representative retinas from CCN1+/+ and EC-ΔCCN1 mice. **, P < 0.003 versus CCN1+/+ mice (n = 4) of the same age. (I and J) Vascular fronts of flat-mounted IB4-stained CCN1+/+ and EC-ΔCCN1 mouse retinas. IB4 staining was overexposed to visualize tip cell filopodia (arrows). Tip cells were counted in four equivalent areas of each retina. *, P < 0.05 versus CCN1+/+ retinas (n = 6). (K) Quantitative analysis of EC proliferation, as determined by BrdU incorporation. Equivalent areas of CCN1+/+ and EC-ΔCCN1 mouse retinas were compared. Data are means plus SE. *, P < 0.05 versus CCN1+/+ retinas (n = 4). (L) Relative mRNA levels of YAP target genes in retinal lysates of CCN1+/+ and EC-ΔCCN1 mice. Data are means ± SE (n = 3).

Article Snippet: The antibodies used in this study were as follows: polyclonal anti-CCN1 antibody (1:250; Biovision Inc.), anti-YAP (1:250 for immunofluorescence; Santa Cruz), anti-YAP (1:500 for Western blotting; Cell Signaling), anti-YAP (1:100 for ChIP; Active Motif), pYAP-S127 (which recognizes pYAP-S112; 1:500; Cell Signaling), anti-collagen IV (1:500; Developmental Studies Hybridoma Bank), anti-MLK1/MRTF-A (1:100; a generous gift from R. Prywes), anti-MLK1/MRTF-A (1:150; Santa Cruz Biotech), anti-TEAD1 (Santa Cruz Biotech), anti-pAkt (1:250; Cell Signaling), anti-Src kinase (1:250; Fisher Scientific), anti-GAPDH (anti-glyceraldehyde-3-phosphate dehydrogenase; 1:1,000; Santa Cruz Biotech), anti-BrdU–Alexa Fluor 488 (1:500; Molecular Probes), and fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG and tetramethyl rhodamine isocyanate-conjugated goat anti-rabbit IgG antibodies (1:1,000; Vector Laboratories). siRNAs targeting gelsolin and CapZ were obtained from Qiagen.

Techniques: Activation Assay, Expressing, Staining, BrdU Incorporation Assay

Journal: Molecular and Cellular Biology

Article Title: CCN1–Yes-Associated Protein Feedback Loop Regulates Physiological and Pathological Angiogenesis

doi: 10.1128/MCB.00107-19

Figure Lengend Snippet: Active YAP induces CCN1 gene transactivation in HRECs. (A) Schematic layout of the CCN1 promoter with the relative location of the CArG and TEAD consensus sequences in the mouse genome and their conservation in human and mouse. dist, distal; prox, proximal. (B and C) Protein levels of CCN1, pYAP, YAP, TEAD1, and GAPDH. (B) Protein levels were determined by immunoblotting after cell treatment with jasplakinolide (Jasp.; 0.1 μM). (C) Densitometric analysis of the protein bands. Values are means plus SE (n = 3). To facilitate comparisons, CCN1 protein levels in control nontreated cells were set to 1 and those of pYAP were set to 5. *, P < 0.004 versus the value at time zero. au, arbitrary units. (D) Immunostaining of control and jasplakinolide-stimulated cells with YAP antibody. Cell nuclei were stained with DAPI (as shown in panel b for panel a and as shown in panel d for panel c). (E) CCN1 promoter reporter activity in jasplakinolide (Jaspl.)-treated HRECs for 1 h (n = 3). (F and G) YAP and MRTF-A enrichment of the endogenous CCN1 promoter determined by a quantitative ChIP assay in control and jasplakinolide-treated cells. Values are means ± half of the range from three experiments using different cell preparations.

Article Snippet: The antibodies used in this study were as follows: polyclonal anti-CCN1 antibody (1:250; Biovision Inc.), anti-YAP (1:250 for immunofluorescence; Santa Cruz), anti-YAP (1:500 for Western blotting; Cell Signaling), anti-YAP (1:100 for ChIP; Active Motif), pYAP-S127 (which recognizes pYAP-S112; 1:500; Cell Signaling), anti-collagen IV (1:500; Developmental Studies Hybridoma Bank), anti-MLK1/MRTF-A (1:100; a generous gift from R. Prywes), anti-MLK1/MRTF-A (1:150; Santa Cruz Biotech), anti-TEAD1 (Santa Cruz Biotech), anti-pAkt (1:250; Cell Signaling), anti-Src kinase (1:250; Fisher Scientific), anti-GAPDH (anti-glyceraldehyde-3-phosphate dehydrogenase; 1:1,000; Santa Cruz Biotech), anti-BrdU–Alexa Fluor 488 (1:500; Molecular Probes), and fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG and tetramethyl rhodamine isocyanate-conjugated goat anti-rabbit IgG antibodies (1:1,000; Vector Laboratories). siRNAs targeting gelsolin and CapZ were obtained from Qiagen.

Techniques: Western Blot, Immunostaining, Staining, Activity Assay

Journal: Molecular and Cellular Biology

Article Title: CCN1–Yes-Associated Protein Feedback Loop Regulates Physiological and Pathological Angiogenesis

doi: 10.1128/MCB.00107-19

Figure Lengend Snippet: YAP-dependent transactivation of the CCN1 gene during retinal vascular development. (A) CCN1 mRNA levels determined by qPCR in mouse retinas at P3, P7, and P14. Data shown are means ± SE (n = 4). (B to D) YAP enrichment of the endogenous CCN1 promoter determined by ChIP assay. DNA was immunoprecipitated from retinal homogenates pooled from 10 mouse eyes of each genotype (YAP+/+, EC-ΔYAP, and UB-ΔYAP). YAP- and MRTF-A-immunoprecipitated DNA was quantified by qPCR. (E) Schematic diagram showing conditional targeting of the YAP genomic locus with Cdh5-Cre ERT2 and UBC-Cre ERT2 mice. (F and G) YAP enrichment of the proximal TEAD site of the endogenous CCN1 gene was analyzed by ChIP assay. (H) Representative immunofluorescence images of IB4-stained retinal flat mounts at P6 of YAP+/+, EC-ΔYAP, and UB-ΔYAP mice. Vascular features were analyzed with AngioTool software (as shown in panel b for panel a, as shown in panel d for panel c, and as shown in panel f for panel e). The outline of the vasculature is shown in black, the vasculature skeleton representation is shown in red (top row), and branching points are shown in green (bottom row). (I to K) Quantitative analysis of vascular parameters (i.e., vascular area [I], junction density [J]) of representative retinas from wild-type and mutant mice. **, P < 0.05 versus YAP+/+ mice (n = 4) of the same age. (K) Quantitative analysis of EC proliferation at P6, as determined by BrdU incorporation. Equivalent areas of retinas from YAP+/+, EC-ΔYAP, and UB-ΔYAP mice were compared. Data are means ± SE. *, P < 0.05 versus YAP+/+ mice; **, P < 0.001 versus YAP+/+ mice (n = 4). (L) Quantification of TUNEL-positive cells in retinal sections of wild-type and YAP mutant mice (n = 6 for each group). Data are presented as means ± SE (n = 6).

Article Snippet: The antibodies used in this study were as follows: polyclonal anti-CCN1 antibody (1:250; Biovision Inc.), anti-YAP (1:250 for immunofluorescence; Santa Cruz), anti-YAP (1:500 for Western blotting; Cell Signaling), anti-YAP (1:100 for ChIP; Active Motif), pYAP-S127 (which recognizes pYAP-S112; 1:500; Cell Signaling), anti-collagen IV (1:500; Developmental Studies Hybridoma Bank), anti-MLK1/MRTF-A (1:100; a generous gift from R. Prywes), anti-MLK1/MRTF-A (1:150; Santa Cruz Biotech), anti-TEAD1 (Santa Cruz Biotech), anti-pAkt (1:250; Cell Signaling), anti-Src kinase (1:250; Fisher Scientific), anti-GAPDH (anti-glyceraldehyde-3-phosphate dehydrogenase; 1:1,000; Santa Cruz Biotech), anti-BrdU–Alexa Fluor 488 (1:500; Molecular Probes), and fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG and tetramethyl rhodamine isocyanate-conjugated goat anti-rabbit IgG antibodies (1:1,000; Vector Laboratories). siRNAs targeting gelsolin and CapZ were obtained from Qiagen.

Techniques: Immunoprecipitation, Immunofluorescence, Staining, Software, Mutagenesis, BrdU Incorporation Assay, TUNEL Assay

Journal: Molecular and Cellular Biology

Article Title: CCN1–Yes-Associated Protein Feedback Loop Regulates Physiological and Pathological Angiogenesis

doi: 10.1128/MCB.00107-19

Figure Lengend Snippet: Expression of CCN1 controls YAP cellular localization and activity in HRECs. (A) Expression of YAP and pYAP in HRECs expressing increasing concentrations of Ad-CCN1 (MOI, 1 to 5), as determined by Western blotting and densitometric scanning of protein bands. (B) Cytoplasmic localization of YAP in HRECs overexpressing CCN1. Cells were transduced with either Ad-luc or Ad-CCN1 (3 MOI) and further transfected with GFP-tagged YAP (i.e., pEGFP-C3-hYAP1). (C and D) Expression of CCN1, YAP, and pYAP in HRECs transduced with Ad-CCN1 and exposed to VEGF (50 ng/ml) for 4 h. *, P < 0.0003 (n = 3). (E) Effects of CCN1 on YAP transcriptional activity, as determined upon cell transfection with the 8×GTIIC-lux plasmid. *, P < 0.001 (n = 3). (F) Effect of function-blocking integrin antibodies (Ab) on CCN1-induced YAP phosphorylation. *, P < 0.0004; **, P < 0.0003 (n = 3). (G and H) Effects of small Rho GTPases and pharmacological inhibitors on CCN1-mediated YAP inactivation. *, P < 0.05 versus conditions with VEGF (n = 3); NS, not significant. (I) Phosphorylation status of potential YAP kinases (e.g., Erk1/2, Akt, and Src kinase) upon VEGF stimulation of CCN1-overexpressing cells. Protein lysates were analyzed by Western immunoblotting with the indicated antibodies. Cntl, control; Veh, vehicle; pYAP, pErk1/2, pAkt, and pSrc kinase, phosphorylated YAP, Erk1/2, Akt, and Src kinase, respectively.

Article Snippet: The antibodies used in this study were as follows: polyclonal anti-CCN1 antibody (1:250; Biovision Inc.), anti-YAP (1:250 for immunofluorescence; Santa Cruz), anti-YAP (1:500 for Western blotting; Cell Signaling), anti-YAP (1:100 for ChIP; Active Motif), pYAP-S127 (which recognizes pYAP-S112; 1:500; Cell Signaling), anti-collagen IV (1:500; Developmental Studies Hybridoma Bank), anti-MLK1/MRTF-A (1:100; a generous gift from R. Prywes), anti-MLK1/MRTF-A (1:150; Santa Cruz Biotech), anti-TEAD1 (Santa Cruz Biotech), anti-pAkt (1:250; Cell Signaling), anti-Src kinase (1:250; Fisher Scientific), anti-GAPDH (anti-glyceraldehyde-3-phosphate dehydrogenase; 1:1,000; Santa Cruz Biotech), anti-BrdU–Alexa Fluor 488 (1:500; Molecular Probes), and fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG and tetramethyl rhodamine isocyanate-conjugated goat anti-rabbit IgG antibodies (1:1,000; Vector Laboratories). siRNAs targeting gelsolin and CapZ were obtained from Qiagen.

Techniques: Expressing, Activity Assay, Western Blot, Transduction, Transfection, Plasmid Preparation, Blocking Assay

Journal: Molecular and Cellular Biology

Article Title: CCN1–Yes-Associated Protein Feedback Loop Regulates Physiological and Pathological Angiogenesis

doi: 10.1128/MCB.00107-19

Figure Lengend Snippet: CCN1 induces YAP phosphorylation through actin capping/severing proteins. (A) Mean spread area of HRECs cultured under the indicated conditions. *, P < 0.05 (n = 20 to 45 cells). (B) Close-up view of YAP (green)- and phalloidin (Phal; red)-stained HRECs following Ad-CCN1 and/or VEGF treatment. The additional effect of substrate stiffness was tested by plating the Ad-CCN1-transduced cells on type I collagen-coated wells. (C) Effects of CCN1 and type I collagen on VEGF-induced YAP activation determined by the 8×GTIIC-lux reporter activity. *, P < 0.003 (n = 4). (D) Effects of formin- and Arp-dependent F-actin inhibitors (SMIFH2 [1, 10, 50 μM] and CK666 [1, 10, 50 μM], respectively) on CCN1-dependent regulation of YAP activity determined by the 8×GTIIC-lux reporter activity (n = 4). (E and F) Depletion of CapZ or gelsolin with siRNAs rescued YAP inhibition by CCN1. (E) Phosphorylation of YAP was analyzed by Western blotting upon transfection of the cells with CapZ or gelsolin siRNAs. (F) Densitometric measurement of the protein bands. *, P < 0.004 (n = 3). (G) IB4-stained retinal flat mounts of P5 mice upon intravitreous injection of the SMIFH2 inhibitor (1 μl, 10 μM) or gelsolin siRNA. (H) A representative image of the phosphorylation pattern of YAP in retinal lysates analyzed by Western blotting upon SMIFH2 inhibitor or gelsolin siRNA injection. Animal treatment was as described in the legend to panel G. siCtrl, siCpz, and siGlsn or siGln, control, Cpz, and gelsolin siRNAs, respectively.

Article Snippet: The antibodies used in this study were as follows: polyclonal anti-CCN1 antibody (1:250; Biovision Inc.), anti-YAP (1:250 for immunofluorescence; Santa Cruz), anti-YAP (1:500 for Western blotting; Cell Signaling), anti-YAP (1:100 for ChIP; Active Motif), pYAP-S127 (which recognizes pYAP-S112; 1:500; Cell Signaling), anti-collagen IV (1:500; Developmental Studies Hybridoma Bank), anti-MLK1/MRTF-A (1:100; a generous gift from R. Prywes), anti-MLK1/MRTF-A (1:150; Santa Cruz Biotech), anti-TEAD1 (Santa Cruz Biotech), anti-pAkt (1:250; Cell Signaling), anti-Src kinase (1:250; Fisher Scientific), anti-GAPDH (anti-glyceraldehyde-3-phosphate dehydrogenase; 1:1,000; Santa Cruz Biotech), anti-BrdU–Alexa Fluor 488 (1:500; Molecular Probes), and fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG and tetramethyl rhodamine isocyanate-conjugated goat anti-rabbit IgG antibodies (1:1,000; Vector Laboratories). siRNAs targeting gelsolin and CapZ were obtained from Qiagen.

Techniques: Cell Culture, Staining, Activation Assay, Activity Assay, Inhibition, Western Blot, Transfection, Injection

Journal: Molecular and Cellular Biology

Article Title: CCN1–Yes-Associated Protein Feedback Loop Regulates Physiological and Pathological Angiogenesis

doi: 10.1128/MCB.00107-19

Figure Lengend Snippet: AAV-mediated expression of CCN1-induced YAP phosphorylation and reduced neovascular growth in retinas with OIR. (A and B) Representative flat-mount preparations of IB4-stained retinas from control and OIR mice at P12 and P17. (C and D) Flat-mount images of IB4-stained retinas from OIR mice at P17 following retro-orbital injection of AAV6-GFP, AAV6–dn-YAP, or AAV6-CCN1. (E) Percentage of neovascular tufts determined by computer-assisted image analysis. **, P < 0.05 versus conditions with AAV6-GFP. (F) Quantitative analysis of CCN1 mRNA levels in the retinas of OIR mice that were injected with AAV6-GFP, AAV6–dn-YAP, or AAV6-CCN1. Data are means ± SE. *, P < 0.05 versus conditions with AAV6-GFP; **, P < 0.001 versus conditions with AAV6-GFP. (G) Qualitative analysis of CCN1, pYAP, and YAP proteins by Western immunoblotting in retinal lysates from OIR mice following retro-orbital injection at P4 of either AAV6-GFP, AAV6–dn-YAP, or AAV6-CCN1. (H) Relative mRNA levels of VEGF-A in retinal lysates of the AAV6-GFP, AAV6–dn-YAP, or AAV6-CCN1 mouse groups.

Article Snippet: The antibodies used in this study were as follows: polyclonal anti-CCN1 antibody (1:250; Biovision Inc.), anti-YAP (1:250 for immunofluorescence; Santa Cruz), anti-YAP (1:500 for Western blotting; Cell Signaling), anti-YAP (1:100 for ChIP; Active Motif), pYAP-S127 (which recognizes pYAP-S112; 1:500; Cell Signaling), anti-collagen IV (1:500; Developmental Studies Hybridoma Bank), anti-MLK1/MRTF-A (1:100; a generous gift from R. Prywes), anti-MLK1/MRTF-A (1:150; Santa Cruz Biotech), anti-TEAD1 (Santa Cruz Biotech), anti-pAkt (1:250; Cell Signaling), anti-Src kinase (1:250; Fisher Scientific), anti-GAPDH (anti-glyceraldehyde-3-phosphate dehydrogenase; 1:1,000; Santa Cruz Biotech), anti-BrdU–Alexa Fluor 488 (1:500; Molecular Probes), and fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG and tetramethyl rhodamine isocyanate-conjugated goat anti-rabbit IgG antibodies (1:1,000; Vector Laboratories). siRNAs targeting gelsolin and CapZ were obtained from Qiagen.

Techniques: Expressing, Staining, Injection, Western Blot