Journal: Stem Cells International

Article Title: Bone Marrow-Derived CD44 + Cells Migrate to Tissue-Engineered Constructs via SDF-1/CXCR4-JNK Pathway and Aid Bone Repair

doi: 10.1155/2019/1513526

Figure Lengend Snippet: Primers used for RT-PCR.

Article Snippet: Arp2/3 Ab , Western blot , 0.1 μ g/ml , 24 h , CST, USA.

Techniques: Sequencing

Journal: Stem Cells International

Article Title: Bone Marrow-Derived CD44 + Cells Migrate to Tissue-Engineered Constructs via SDF-1/CXCR4-JNK Pathway and Aid Bone Repair

doi: 10.1155/2019/1513526

Figure Lengend Snippet: Reagents used for in vitro experiments.

Article Snippet: Arp2/3 Ab , Western blot , 0.1 μ g/ml , 24 h , CST, USA.

Techniques: In Vitro, Concentration Assay, FACS, Flow Cytometry, Immunofluorescence, Western Blot

Journal: Stem Cells International

Article Title: Bone Marrow-Derived CD44 + Cells Migrate to Tissue-Engineered Constructs via SDF-1/CXCR4-JNK Pathway and Aid Bone Repair

doi: 10.1155/2019/1513526

Figure Lengend Snippet: JNK served as an effector downstream of SDF-1/CXCR4 in the migration of bone marrow (BM) CD44 + cells towards tissue-engineered constructs (TECs). (a) Comparison of phosphorylated JNK (p-JNK), ERK (p-ERK), and P38 (p-P38) expression in BM cells after CXCR4 blockade. After migration stopped, BM cells were collected and analyzed by western blot. (b) Representative images of cell migration towards SDF-1. The quantification of migrated BM cells is shown as a bar graph ( n = 5). ∗∗ P < 0.01. (c) FACS analysis of the proportions of CD44 + cells in peripheral blood (PB). The quantification comparison is shown as a bar graph ( n = 3). ∗∗ P < 0.01. (d) Representative images of in vivo migration of GFP + /CD44 + cells towards TECs. The introduction of SP600125 significantly reduced the recruitment of GFP + /CD44 + cells. White triangle, implant area; white arrows, CD44 + cells; scale bar, 50 mm. (e) Comparison of Arpin and Arp2/3 expressions in BM cells after JNK blockade in vitro . (f) Analysis of the Arpin, Arp2, and Arp3 mRNA expression in sorted CD44 + cells. On day 3 postoperatively, cells in PB were sorted by FACS on CD44. RT-PCR was performed to evaluate Arpin, Arp2, and Arp3 mRNA expression. The quantification data are shown as a bar graph ( n = 3). ∗∗ P < 0.01.

Article Snippet: Arp2/3 Ab , Western blot , 0.1 μ g/ml , 24 h , CST, USA.

Techniques: Migration, Construct, Expressing, Western Blot, In Vivo, In Vitro, Reverse Transcription Polymerase Chain Reaction

Journal: Stem Cells International

Article Title: Bone Marrow-Derived CD44 + Cells Migrate to Tissue-Engineered Constructs via SDF-1/CXCR4-JNK Pathway and Aid Bone Repair

doi: 10.1155/2019/1513526

Figure Lengend Snippet: Primers used for RT-PCR.

Article Snippet: After blocking with 5% milk, membranes were incubated overnight at 4°C with the following primary antibodies: anti-CXCR4, anti-p-ERK, anti-p38, anti-p-JNK, and anti-Arpin (1 : 1000 dilution; Abcam, USA), and anti-Arp2/3 (1 : 1000 dilution; CST, USA), followed by incubation with horseradish peroxidase-conjugated secondary antibody (1 : 2000 dilution; SouthernBiotech, Birmingham, AL) at room temperature for 1 hour.

Techniques: Sequencing

Journal: Stem Cells International

Article Title: Bone Marrow-Derived CD44 + Cells Migrate to Tissue-Engineered Constructs via SDF-1/CXCR4-JNK Pathway and Aid Bone Repair

doi: 10.1155/2019/1513526

Figure Lengend Snippet: Reagents used for in vitro experiments.

Article Snippet: After blocking with 5% milk, membranes were incubated overnight at 4°C with the following primary antibodies: anti-CXCR4, anti-p-ERK, anti-p38, anti-p-JNK, and anti-Arpin (1 : 1000 dilution; Abcam, USA), and anti-Arp2/3 (1 : 1000 dilution; CST, USA), followed by incubation with horseradish peroxidase-conjugated secondary antibody (1 : 2000 dilution; SouthernBiotech, Birmingham, AL) at room temperature for 1 hour.

Techniques: In Vitro, Concentration Assay, FACS, Flow Cytometry, Immunofluorescence, Western Blot

Journal: Stem Cells International

Article Title: Bone Marrow-Derived CD44 + Cells Migrate to Tissue-Engineered Constructs via SDF-1/CXCR4-JNK Pathway and Aid Bone Repair

doi: 10.1155/2019/1513526

Figure Lengend Snippet: JNK served as an effector downstream of SDF-1/CXCR4 in the migration of bone marrow (BM) CD44 + cells towards tissue-engineered constructs (TECs). (a) Comparison of phosphorylated JNK (p-JNK), ERK (p-ERK), and P38 (p-P38) expression in BM cells after CXCR4 blockade. After migration stopped, BM cells were collected and analyzed by western blot. (b) Representative images of cell migration towards SDF-1. The quantification of migrated BM cells is shown as a bar graph ( n = 5). ∗∗ P < 0.01. (c) FACS analysis of the proportions of CD44 + cells in peripheral blood (PB). The quantification comparison is shown as a bar graph ( n = 3). ∗∗ P < 0.01. (d) Representative images of in vivo migration of GFP + /CD44 + cells towards TECs. The introduction of SP600125 significantly reduced the recruitment of GFP + /CD44 + cells. White triangle, implant area; white arrows, CD44 + cells; scale bar, 50 mm. (e) Comparison of Arpin and Arp2/3 expressions in BM cells after JNK blockade in vitro . (f) Analysis of the Arpin, Arp2, and Arp3 mRNA expression in sorted CD44 + cells. On day 3 postoperatively, cells in PB were sorted by FACS on CD44. RT-PCR was performed to evaluate Arpin, Arp2, and Arp3 mRNA expression. The quantification data are shown as a bar graph ( n = 3). ∗∗ P < 0.01.

Article Snippet: After blocking with 5% milk, membranes were incubated overnight at 4°C with the following primary antibodies: anti-CXCR4, anti-p-ERK, anti-p38, anti-p-JNK, and anti-Arpin (1 : 1000 dilution; Abcam, USA), and anti-Arp2/3 (1 : 1000 dilution; CST, USA), followed by incubation with horseradish peroxidase-conjugated secondary antibody (1 : 2000 dilution; SouthernBiotech, Birmingham, AL) at room temperature for 1 hour.

Techniques: Migration, Construct, Expressing, Western Blot, In Vivo, In Vitro, Reverse Transcription Polymerase Chain Reaction

Journal: Journal of Cancer

Article Title: Antimetastatic Effect of Fucoidan-Sargassum against Liver Cancer Cell Invadopodia Formation via Targeting Integrin αVβ3 and Mediating αVβ3/Src/E2F1 Signaling

doi: 10.7150/jca.26740

Figure Lengend Snippet: Fucoidan-Sargassum inhibits the initiation and assembly stages of invadopodia formation in HCCLM3 cells. (A) The elongated protrusions of the HCCLM3 cells, which are called invadopodia; the image was taken under an inverted microscope (400× magnification). (B) Western blotting analysis of FAK, Src, and Cortactin, which are key proteins involved in the initiation stage of invadopodia formation, after the cells were treated with Fucoidan-Sargassum (0, 10, 20, and 30 mg/mL) for 48 h. (C) Confocal images of fixed control or Fucoidan-Sargassum-treated cells immunostained for actin (2nd panel), cortactin (middle panel), and Src (4th panel). The arrowheads indicate active invadopodia initiation in the control cells and the cytoskeletal changes in the treated cells. Scale bars: 7.5 µm. (D and E) Western blotting analysis of TKS5, N-WASP, ARP3, ARP2, and CDC42, which are key proteins involved in the assembly stage of invadopodia formation, and MMP2 and MT1-MMP, which are involved in the maturation stage of invadopodia formation, after the cells were treated with Fucoidan-Sargassum (0, 10, 20, and 30 mg/mL) for 48 h.

Article Snippet: Rabbit polyclonal antibodies were used: anti-Tublin, anti-IRE1a were from Beyotime Biotechnology (Shanghai, China), anti-TKS5, anti-ITGα1 were from Zen-Biotechnology (Sichuan, China), anti-GRP78, aniti-GRP94, anti-SPARC, anti-c-Src were from Proteintech (Rosemont, IL, USA), anti-ITGαV, anti-ITGβ3, anti-ITGβ1, anti-Src, anti-phospho-Src416, anti-phospho-cortactin, anti-FAK, anti-phospho-FAK, anti-ARP2, anti-ARP3, anti-N-WASP, anti-WAVE-2, anti-Rac1/Cdc42, anti-phospho-Rac1/Cdc42(ser71), anti-E2F1, anti-phospho -E2F1, anti-phospho-MPZL1 were from Cell Signaling Technology (Danvers, MA, USA), anti-MPZL was from Abcam (Cambridge, MA, USA).

Techniques: Inverted Microscopy, Western Blot

Journal: PLoS Pathogens

Article Title: Neisseria gonorrhoeae subverts formin-dependent actin polymerization to colonize human macrophages

doi: 10.1371/journal.ppat.1010184

Figure Lengend Snippet: (A-E) Gonococci internalization by U937 MFs (A-D, H and J) and hMDMs (E) requires formin-mediated actin polymerization but not canonical phagocytosis. (A-E, H and J) Quantitative analysis of bacteria uptake by macrophages using inside/out microscopy in the presence/absence of different inhibitors added 30 min prior to infection. Gonococcal infections were stopped at 8hpi and the percentage of intracellular colonies in each condition was calculated (green bars). L . pneumophila ΔdotA infections were stopped at 2hpi and the percentage of internalized bacteria was determined (gray bars). The internalization index reflects how each treatment affects uptake compared to vehicle control and was calculated for each condition by dividing the percentage of internalized objects from the treatment condition by the percentage of internalized objects from the respective vehicle control condition, which was then multiplied by 100. Inhibitors targeting: (A) actin polymerization (cytochalasin D, 5μM) and microtubules polymerization (nocodazole, 3μM); (B) the small GTPases Cdc42 (ML-141, 5μM), Rac1/2/3 (EHT 1864, 5μM) and the downstream effectors of Rho GTPase—LIMK1/2 kinases (BMS-5, 1μM) and ROCK1/2 kinases (GSK269962, 1μM); (C) the phagosome regulators Dynamin (dynasore, 30μM) and Phosphoinositide-3 kinase (LY294002, 10μM); (D) actin polymerization regulators formins (SMIFH2, 12.5 or 25μM), Arp2/3 complex (CK869, 10μM), Myosin II ATPases ((-) blebbistatin, from 2.5 to 20μM). (E) Decreased gonococci uptake at 8hpi by hMDMs treated with SMIFH2 as indicated. (F) Immunoblot analysis of the U937 cell line stably expressing Cas9 used for CRISPR genome editing. (G) Immunoblot validation for the loss of Arp2 in the ACTR2 KO and LC3 in the MAP1LC3B KO U937 MFs. (H and J) N . g colony internalization is not affected by the loss of (H) Arp2 or (J) LC3. (I) mRNA expression for the indicated genes in U937 cells from RNAseq presented in normalized transcript per million (nTPMs). Data obtained from the Human Atlas Project : MAP1LC3A ( https://www.proteinatlas.org/ENSG00000101460-MAP1LC3A/cell+line ); MAP1LC3B ( https://www.proteinatlas.org/ENSG00000140941-MAP1LC3B/cell+line ); MAP1LC3C ( https://www.proteinatlas.org/ENSG00000197769-MAP1LC3C/cell+line ). (A-E, H and J) Means from at least three biological replicates ± SD. At least 100 N . g colonies or L . p ΔdotA bacteria for each condition were scored. The statistical significance of the differences between the internalization index from inhibitor-treated cells and the vehicle-treated cells for each condition were calculated using the unpaired T-test (A-C, D for Arp2/3INH, H and J) or one-way ANOVA (D for Formin INH and MyosinII INH , E) , not significant (ns.).

Article Snippet: Rabbit monoclonal antibodies against the following antigens were purchased from Cell Signaling—Rab5 (C8B1), Rab7 (D95F2), EEA1 (C45B10), GM130 (D6B1), Ezrin (#3145), LC3A/B (D3U4C), V5-Tag (E9H80), CEACAM1 (D3R80) and ACTR2 (#3128S).

Techniques: Microscopy, Infection, Western Blot, Stable Transfection, Expressing, CRISPR

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: MFG‐E8 Regulates Vascular Smooth Muscle Cell Migration Through Dose‐Dependent Mediation of Actin Polymerization

doi: 10.1161/JAHA.121.020870

Figure Lengend Snippet: A and B , Representative immunofluorescence photographs of Arp2 ( A ) and actin‐related protein 3 (Arp3) ( B ) in the carotid arteries of wild‐type (WT) and MFG‐E8 knockout (KO) mice. Bar, 50 μm. Quantification of fluorescence intensities of Arp2 (WT: n mice =5, MFG‐E8 knockout: n mice =5) and Arp3 (WT: n mice =7, MFG‐E8 knockout: n mice =7) is shown in ( A ) and ( B ), respectively. Results are presented as mean±SEM. Each point is derived from an assessment of 3 sections of an individual animal. **** P <0.0001, as obtained using the t test. C , Proteins were extracted from 10 pooled common carotid arteries of WT and knockout mice for Western blotting and examined with antibodies against Arp2 and Arp3. In the quantitative analysis, the Arp2 and Arp3 levels were normalized to that of GAPDH. D , A10 VSMCs underwent 48‐hour transfection with small interfering RNA (siRNA) against MFG‐E8 (si‐MFG‐E8) or control siRNA (Ctrl siRNA). The expression of Arp2 and Arp3 was evaluated through Western blotting. The Arp2 (n=3) and Arp3 (n=3) levels were normalized to those of α‐tubulin. Three repeated experiments were performed. Data are presented as mean±SD. Each point is derived from each of the 3 repeated experiments. ** P <0.01, as obtained using the t test. CCA indicates common carotid artery; and DAPI, 4',6‐diamidino‐2‐phenylindole.

Article Snippet: To determine the expression of Arp2 and Arp3 in the mouse CCAs, the paraffinized sections within the first millimeter proximal to the ligature were deparaffinized, permeabilized with 0.1% Triton‐X 100 in PBS for 10 minutes, blocked with 5% normal goat serum in 0.1% Triton‐X 100 in PBS, and incubated with primary antibodies against Arp2 and Arp3 (1:200, Cell Signaling) overnight at 4°C; this was followed by an additional incubation with Alexa Fluor 594–conjugated secondary antibodies (1:100, Thermo Fisher Invitrogen) for 1 hour at room temperature.

Techniques: Immunofluorescence, Knock-Out, Fluorescence, Derivative Assay, Western Blot, Transfection, Small Interfering RNA, Expressing

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: MFG‐E8 Regulates Vascular Smooth Muscle Cell Migration Through Dose‐Dependent Mediation of Actin Polymerization

doi: 10.1161/JAHA.121.020870

Figure Lengend Snippet: A through C , A10 VSMCs were treated with either a low (10 ng/mL) or high (1000 ng/mL) concentration of recombinant MFG‐E8 for 16 hours. A , Western blot analysis of the protein expression of Arp2 and actin‐related protein (Arp3) in A10 cells treated with low and high doses of MFG‐E8 for 16 hours. Quantitative analyses of Arp2 ( B ) and Arp3 ( C ) levels normalized to those of α‐tubulin were conducted (n=3). Data are presented as mean±SD. Three independent experiments were performed. Each point is derived from each of the 3 repeated experiments. * P <0.05, ** P <0.01, *** P <0.001, as obtained using 1‐way ANOVA followed by Tukey multiple comparisons test. D through E , VSMCs isolated from aortas of wild‐type mice were immunostained with antibodies against Arp2 ( D ) and Arp3 ( E ) and costained with phalloidin to label filamentous actin (F‐actin). Bar, 50 μm. Three independent experiments were performed and each experiment was repeated with similar results. In a representative experiment, the fluorescence intensities of Arp2 (n=3–5) ( D ) and Arp3 (n=3) ( E ) were quantitated. Data are presented as mean±SD. Each point is derived from an assessment of each picture field. * P <0.05 and **** P <0.0001, as obtained using 1‐way ANOVA followed by Tukey multiple comparisons test. DAPI indicates 4',6‐diamidino‐2‐phenylindole.

Article Snippet: To determine the expression of Arp2 and Arp3 in the mouse CCAs, the paraffinized sections within the first millimeter proximal to the ligature were deparaffinized, permeabilized with 0.1% Triton‐X 100 in PBS for 10 minutes, blocked with 5% normal goat serum in 0.1% Triton‐X 100 in PBS, and incubated with primary antibodies against Arp2 and Arp3 (1:200, Cell Signaling) overnight at 4°C; this was followed by an additional incubation with Alexa Fluor 594–conjugated secondary antibodies (1:100, Thermo Fisher Invitrogen) for 1 hour at room temperature.

Techniques: Concentration Assay, Recombinant, Western Blot, Expressing, Derivative Assay, Isolation, Fluorescence

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: MFG‐E8 Regulates Vascular Smooth Muscle Cell Migration Through Dose‐Dependent Mediation of Actin Polymerization

doi: 10.1161/JAHA.121.020870

Figure Lengend Snippet: A through C , A10 vascular smooth muscle cells (VSMCs) incubated with either 50 μg/mL β1 inhibitory antibody (Itgb1 Ab) or isotype control 4 hours before treatment with either a low or high concentration of recombinant MFG‐E8 for 16 hours. A , Immunoblotting was performed to assess the protein expression of Arp2 and actin‐related protein 3 (Arp3) in A10 cells in a representative experiment. Quantitative analyses of Arp2 ( B ) and Arp3 ( C ) normalized to GAPDH were conducted (n=3). Data are presented as mean±SD. Three repeated experiments were performed. Each point is derived from each of the 3 repeated experiments. * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001, as obtained using 1‐way ANOVA followed by Tukey multiple comparisons test. D , Representative confocal fluorescent images of phalloidin for filamentous actin (F‐actin) (green) and DNase I for globular actin (G‐actin) (red) staining of A10 VSMCs stimulated with low (10 ng/mL) and high (1000 ng/mL) doses of MFG‐E8 for 16 hours. Bar, 50 μm. In a representative experiment, corresponding quantification of the mean fluorescence intensity of phalloidin for F‐actin (n=5) and that of DNase I for G‐actin (n=4–6) was performed. Data are presented as mean±SD. Each point is derived from an assessment of each picture field. ** P <0.01 and **** P <0.0001, as obtained using 1‐way ANOVA followed by Tukey multiple comparisons test.

Article Snippet: To determine the expression of Arp2 and Arp3 in the mouse CCAs, the paraffinized sections within the first millimeter proximal to the ligature were deparaffinized, permeabilized with 0.1% Triton‐X 100 in PBS for 10 minutes, blocked with 5% normal goat serum in 0.1% Triton‐X 100 in PBS, and incubated with primary antibodies against Arp2 and Arp3 (1:200, Cell Signaling) overnight at 4°C; this was followed by an additional incubation with Alexa Fluor 594–conjugated secondary antibodies (1:100, Thermo Fisher Invitrogen) for 1 hour at room temperature.

Techniques: Incubation, Concentration Assay, Recombinant, Western Blot, Expressing, Derivative Assay, Staining, Fluorescence

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: MFG‐E8 Regulates Vascular Smooth Muscle Cell Migration Through Dose‐Dependent Mediation of Actin Polymerization

doi: 10.1161/JAHA.121.020870

Figure Lengend Snippet: A , A10 vascular smooth muscle cells (VSMCs) incubated with either 50 μg/mL β1 inhibitory antibody (Itgb1 Ab) or isotype control 4 hours before treatment with either a low or high concentration of recombinant MFG‐E8 for 16 hours. Rac1 activity was measured using the G‐LISA (Cytoskeleton) assay. Data represent the averages of duplicate samples (n=2), and the error bars indicate the SD. * P <0.05, ** P <0.01, *** P <0.001, as obtained using 1‐way ANOVA followed by Tukey multiple comparisons test. B and C , A10 cells were incubated with the Rac1 inhibitor NSC23766 (200 μM, Tocris) 16 hours before treatment with low and high doses of MFG‐E8 for 16 hours. B , Immunoblotting analysis of the Arp2 protein expression in A10 VSMCs was performed. C , Quantitative analysis of Arp2 normalized to GAPDH was performed (n=4). Data are presented as mean±SD. Four repeated experiments were performed. Each point is derived from each of the 4 repeated experiments. * P <0.05 and ** P <0.01, as obtained using 1‐way ANOVA followed by Tukey multiple comparisons test.

Article Snippet: To determine the expression of Arp2 and Arp3 in the mouse CCAs, the paraffinized sections within the first millimeter proximal to the ligature were deparaffinized, permeabilized with 0.1% Triton‐X 100 in PBS for 10 minutes, blocked with 5% normal goat serum in 0.1% Triton‐X 100 in PBS, and incubated with primary antibodies against Arp2 and Arp3 (1:200, Cell Signaling) overnight at 4°C; this was followed by an additional incubation with Alexa Fluor 594–conjugated secondary antibodies (1:100, Thermo Fisher Invitrogen) for 1 hour at room temperature.

Techniques: Incubation, Concentration Assay, Recombinant, Activity Assay, Western Blot, Expressing, Derivative Assay

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: MFG‐E8 Regulates Vascular Smooth Muscle Cell Migration Through Dose‐Dependent Mediation of Actin Polymerization

doi: 10.1161/JAHA.121.020870

Figure Lengend Snippet: Common carotid arteries (CCAs) of wild‐type (WT) mice were ligated, and recombinant MFG‐E8 (2 μg/mL) was delivered using pluronic gel into the vessels. A and B , Representative immunofluorescence images display the expression of Arp2 ( A ) and Arp3 ( B ) in the sham‐operated CCAs, ligated CCAs, and ligated CCAs treated with high‐dose MFG‐E8 in WT mice 21 days after ligation. Lumen (L), neointima (I), and media (M) of the vessels are indicated. Bar, 50 μm. C , Protein levels of Arp2 were quantified through immunoblotting. Proteins were extracted from 10 pooled CCAs per group for Western blotting and examined using antibodies against Arp2. In the quantitative analysis, the Arp2 and actin‐related protein 3 (Arp3) levels were normalized to that of β‐tubulin. DAPI indicates 4',6‐diamidino‐2‐phenylindole.

Article Snippet: To determine the expression of Arp2 and Arp3 in the mouse CCAs, the paraffinized sections within the first millimeter proximal to the ligature were deparaffinized, permeabilized with 0.1% Triton‐X 100 in PBS for 10 minutes, blocked with 5% normal goat serum in 0.1% Triton‐X 100 in PBS, and incubated with primary antibodies against Arp2 and Arp3 (1:200, Cell Signaling) overnight at 4°C; this was followed by an additional incubation with Alexa Fluor 594–conjugated secondary antibodies (1:100, Thermo Fisher Invitrogen) for 1 hour at room temperature.

Techniques: Recombinant, Immunofluorescence, Expressing, Ligation, Western Blot

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: MFG‐E8 Regulates Vascular Smooth Muscle Cell Migration Through Dose‐Dependent Mediation of Actin Polymerization

doi: 10.1161/JAHA.121.020870

Figure Lengend Snippet: A and B , Representative immunofluorescence photographs of Arp2 ( A ) and actin‐related protein 3 (Arp3) ( B ) in the carotid arteries of wild‐type (WT) and MFG‐E8 knockout (KO) mice. Bar, 50 μm. Quantification of fluorescence intensities of Arp2 (WT: n mice =5, MFG‐E8 knockout: n mice =5) and Arp3 (WT: n mice =7, MFG‐E8 knockout: n mice =7) is shown in ( A ) and ( B ), respectively. Results are presented as mean±SEM. Each point is derived from an assessment of 3 sections of an individual animal. **** P <0.0001, as obtained using the t test. C , Proteins were extracted from 10 pooled common carotid arteries of WT and knockout mice for Western blotting and examined with antibodies against Arp2 and Arp3. In the quantitative analysis, the Arp2 and Arp3 levels were normalized to that of GAPDH. D , A10 VSMCs underwent 48‐hour transfection with small interfering RNA (siRNA) against MFG‐E8 (si‐MFG‐E8) or control siRNA (Ctrl siRNA). The expression of Arp2 and Arp3 was evaluated through Western blotting. The Arp2 (n=3) and Arp3 (n=3) levels were normalized to those of α‐tubulin. Three repeated experiments were performed. Data are presented as mean±SD. Each point is derived from each of the 3 repeated experiments. ** P <0.01, as obtained using the t test. CCA indicates common carotid artery; and DAPI, 4',6‐diamidino‐2‐phenylindole.

Article Snippet: Primary antibodies of Arp2 and Arp3 (Cell Signaling), MFG‐E8 (R&D), and GAPDH or α‐tubulin (Sigma‐Aldrich) were employed for immunoblot analyses, as previously described., Quantitative analysis was performed through densitometry by using ImageJ (National Institutes of Health).

Techniques: Immunofluorescence, Knock-Out, Fluorescence, Derivative Assay, Western Blot, Transfection, Small Interfering RNA, Expressing

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: MFG‐E8 Regulates Vascular Smooth Muscle Cell Migration Through Dose‐Dependent Mediation of Actin Polymerization

doi: 10.1161/JAHA.121.020870

Figure Lengend Snippet: A through C , A10 VSMCs were treated with either a low (10 ng/mL) or high (1000 ng/mL) concentration of recombinant MFG‐E8 for 16 hours. A , Western blot analysis of the protein expression of Arp2 and actin‐related protein (Arp3) in A10 cells treated with low and high doses of MFG‐E8 for 16 hours. Quantitative analyses of Arp2 ( B ) and Arp3 ( C ) levels normalized to those of α‐tubulin were conducted (n=3). Data are presented as mean±SD. Three independent experiments were performed. Each point is derived from each of the 3 repeated experiments. * P <0.05, ** P <0.01, *** P <0.001, as obtained using 1‐way ANOVA followed by Tukey multiple comparisons test. D through E , VSMCs isolated from aortas of wild‐type mice were immunostained with antibodies against Arp2 ( D ) and Arp3 ( E ) and costained with phalloidin to label filamentous actin (F‐actin). Bar, 50 μm. Three independent experiments were performed and each experiment was repeated with similar results. In a representative experiment, the fluorescence intensities of Arp2 (n=3–5) ( D ) and Arp3 (n=3) ( E ) were quantitated. Data are presented as mean±SD. Each point is derived from an assessment of each picture field. * P <0.05 and **** P <0.0001, as obtained using 1‐way ANOVA followed by Tukey multiple comparisons test. DAPI indicates 4',6‐diamidino‐2‐phenylindole.

Article Snippet: Primary antibodies of Arp2 and Arp3 (Cell Signaling), MFG‐E8 (R&D), and GAPDH or α‐tubulin (Sigma‐Aldrich) were employed for immunoblot analyses, as previously described., Quantitative analysis was performed through densitometry by using ImageJ (National Institutes of Health).

Techniques: Concentration Assay, Recombinant, Western Blot, Expressing, Derivative Assay, Isolation, Fluorescence

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: MFG‐E8 Regulates Vascular Smooth Muscle Cell Migration Through Dose‐Dependent Mediation of Actin Polymerization

doi: 10.1161/JAHA.121.020870

Figure Lengend Snippet: A through C , A10 vascular smooth muscle cells (VSMCs) incubated with either 50 μg/mL β1 inhibitory antibody (Itgb1 Ab) or isotype control 4 hours before treatment with either a low or high concentration of recombinant MFG‐E8 for 16 hours. A , Immunoblotting was performed to assess the protein expression of Arp2 and actin‐related protein 3 (Arp3) in A10 cells in a representative experiment. Quantitative analyses of Arp2 ( B ) and Arp3 ( C ) normalized to GAPDH were conducted (n=3). Data are presented as mean±SD. Three repeated experiments were performed. Each point is derived from each of the 3 repeated experiments. * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001, as obtained using 1‐way ANOVA followed by Tukey multiple comparisons test. D , Representative confocal fluorescent images of phalloidin for filamentous actin (F‐actin) (green) and DNase I for globular actin (G‐actin) (red) staining of A10 VSMCs stimulated with low (10 ng/mL) and high (1000 ng/mL) doses of MFG‐E8 for 16 hours. Bar, 50 μm. In a representative experiment, corresponding quantification of the mean fluorescence intensity of phalloidin for F‐actin (n=5) and that of DNase I for G‐actin (n=4–6) was performed. Data are presented as mean±SD. Each point is derived from an assessment of each picture field. ** P <0.01 and **** P <0.0001, as obtained using 1‐way ANOVA followed by Tukey multiple comparisons test.

Article Snippet: Primary antibodies of Arp2 and Arp3 (Cell Signaling), MFG‐E8 (R&D), and GAPDH or α‐tubulin (Sigma‐Aldrich) were employed for immunoblot analyses, as previously described., Quantitative analysis was performed through densitometry by using ImageJ (National Institutes of Health).

Techniques: Incubation, Concentration Assay, Recombinant, Western Blot, Expressing, Derivative Assay, Staining, Fluorescence

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: MFG‐E8 Regulates Vascular Smooth Muscle Cell Migration Through Dose‐Dependent Mediation of Actin Polymerization

doi: 10.1161/JAHA.121.020870

Figure Lengend Snippet: A , A10 vascular smooth muscle cells (VSMCs) incubated with either 50 μg/mL β1 inhibitory antibody (Itgb1 Ab) or isotype control 4 hours before treatment with either a low or high concentration of recombinant MFG‐E8 for 16 hours. Rac1 activity was measured using the G‐LISA (Cytoskeleton) assay. Data represent the averages of duplicate samples (n=2), and the error bars indicate the SD. * P <0.05, ** P <0.01, *** P <0.001, as obtained using 1‐way ANOVA followed by Tukey multiple comparisons test. B and C , A10 cells were incubated with the Rac1 inhibitor NSC23766 (200 μM, Tocris) 16 hours before treatment with low and high doses of MFG‐E8 for 16 hours. B , Immunoblotting analysis of the Arp2 protein expression in A10 VSMCs was performed. C , Quantitative analysis of Arp2 normalized to GAPDH was performed (n=4). Data are presented as mean±SD. Four repeated experiments were performed. Each point is derived from each of the 4 repeated experiments. * P <0.05 and ** P <0.01, as obtained using 1‐way ANOVA followed by Tukey multiple comparisons test.

Article Snippet: Primary antibodies of Arp2 and Arp3 (Cell Signaling), MFG‐E8 (R&D), and GAPDH or α‐tubulin (Sigma‐Aldrich) were employed for immunoblot analyses, as previously described., Quantitative analysis was performed through densitometry by using ImageJ (National Institutes of Health).

Techniques: Incubation, Concentration Assay, Recombinant, Activity Assay, Western Blot, Expressing, Derivative Assay

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: MFG‐E8 Regulates Vascular Smooth Muscle Cell Migration Through Dose‐Dependent Mediation of Actin Polymerization

doi: 10.1161/JAHA.121.020870

Figure Lengend Snippet: Common carotid arteries (CCAs) of wild‐type (WT) mice were ligated, and recombinant MFG‐E8 (2 μg/mL) was delivered using pluronic gel into the vessels. A and B , Representative immunofluorescence images display the expression of Arp2 ( A ) and Arp3 ( B ) in the sham‐operated CCAs, ligated CCAs, and ligated CCAs treated with high‐dose MFG‐E8 in WT mice 21 days after ligation. Lumen (L), neointima (I), and media (M) of the vessels are indicated. Bar, 50 μm. C , Protein levels of Arp2 were quantified through immunoblotting. Proteins were extracted from 10 pooled CCAs per group for Western blotting and examined using antibodies against Arp2. In the quantitative analysis, the Arp2 and actin‐related protein 3 (Arp3) levels were normalized to that of β‐tubulin. DAPI indicates 4',6‐diamidino‐2‐phenylindole.

Article Snippet: Primary antibodies of Arp2 and Arp3 (Cell Signaling), MFG‐E8 (R&D), and GAPDH or α‐tubulin (Sigma‐Aldrich) were employed for immunoblot analyses, as previously described., Quantitative analysis was performed through densitometry by using ImageJ (National Institutes of Health).

Techniques: Recombinant, Immunofluorescence, Expressing, Ligation, Western Blot