Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Self-Triggered Apoptosis Enzyme Prodrug Therapy (STAEPT): Enhancing Targeted Therapies via Recurrent Bystander Killing Effect by Exploiting Caspase-Cleavable Linker.

doi: 10.1002/advs.201800368

Figure Lengend Snippet: Figure 2. Enhanced cellular uptake and cytotoxic activity of RGDEVD-DOX in integrin αvβ3 expressing cells. a) Representative confocal images (left) and quantitative analysis (right) of HDMEC and U-87 MG cells exposed to fluorescent-labeled RDEVD and RGDEVD peptides. b) Representative confocal images (left) and quantitative analysis (right) of scrambled control and integrin αv (ITGAV) siRNA-transfected HDMECs and U-87 MG cells exposed to fluorescent-labeled RGDEVD peptide. Green and blue indicate the fluorescent-labeled peptides and cell nuclei, respectively. Scale bar, 50 µm. c) Flow cytometry analysis of isotype control (top), scrambled control-transfected (lower left), and ITGAV siRNA-transfected (lower right) U87 MG cells incubated with fluorescent-labeled RGDEVD and stained with an antibody against integrin αvβ3. d) Representative confocal images (left) and quantitative analysis (right) of U-87 MG and HT-29 cells treated with RDEVD-DOX and RGDEVD-DOX. Red and blue indicate the intrinsic red fluorescence of doxorubicin and cell nuclei, respectively. Scale bar, 50 µm. e) Concentration-dependent cytotoxicity of RDEVD-DOX and RGDEVD-DOX on U-87 MG (left) and HT-29 (right) determined by MTT assay (n = 4). f) Doxorubicin release from RGDEVD-DOX when incubated in PBS (pH 7.4) containing (or not containing) carboxylesterase (n = 3). Data are mean ± s.d. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: The membranes were blotted with antibodies against procaspase-3 (1:1000; cat. no. 9665), cleaved caspase-3 (1:1000; cat. no. 9664), integrin αv (1:1000; cat. no. 4711), integrin β3 (1:1000; cat. no. 13166), and β-actin (1:2000; cat. no. 3700), which were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Activity Assay, Expressing, Labeling, Control, Transfection, Flow Cytometry, Incubation, Staining, Fluorescence, Concentration Assay, MTT Assay

Journal: Experimental cell research

Article Title: VANGL2 protein stability is regulated by integrin αv and the extracellular matrix.

doi: 10.1016/j.yexcr.2018.11.017

Figure Lengend Snippet: Fig. 1. Integrin αv regulates VANGL2 protein levels but not transcription. (A) Western blot of biotinylated plasma membrane (pm) and cytoplasmic (c) integrin αv (ITGAV) protein from non-targeting (NT) control and single (#10) integrin αv siRNA transfected HT-1080 cells. (B) Western blot of total protein from NT control and integrin αv knockdown cells. #08 is a single siRNA to integrin αv and the pool is a mixture of four integrin αv siRNAs that includes 08 and 10. (C, D) Integrin αv knockdown cells have reduced total VANGL2 protein levels. Box plots show median value ± standard deviation (whiskers) and the interquartile range (boxes) of likely variation (experiment performed three times, n = 6 biological replicates). (E) Quantitative RT-PCR was performed using TaqMan probes for HPRT1 and VANGL2 and cDNA obtained from NT control and integrin αv siRNA transfected cells. Scatter plot of delta Ct values (VANGL2-HPRT1 delta Ct) showing the average value ± standard deviation (experiment performed three times, n = 18 biological replicates). (F) GFP-VANGL2 expression in NT control and integrin αv siRNA transfected cells shown under identical imaging parameters. Arrows denote plasma membrane expression. The integrin αv image was brightened to allow accurate arrow placement and then reduced to the original parameters. *** P < 0.001; **** P < 0.0001; P values are versus NT siRNA in panel D; two-tailed unpaired t-test. Scale bars = 5 µm.

Article Snippet: The primary antibodies were VANGL2 (1:1000, MABN750), Millipore Sigma, St. Louis, MO; integrin αv (1:1000, ab17975), integrin β1 (1:1000, ab30394), and pan-cadherin (1:1000, ab16505) Abcam, Cambridge, MA; integrin α5 (1:1000, #4705), integrin β5 (1:1000, #3629), integrin β3 (1:1000, #13166), and β-actin (1:1000, #4967), Cell Signaling Technology, Danvers, MA; GAPDHHRP (1:2000, GTX627408-01), GeneTex, Inc., Irvine, CA.

Techniques: Western Blot, Clinical Proteomics, Membrane, Control, Transfection, Knockdown, Standard Deviation, Quantitative RT-PCR, Expressing, Imaging, Two Tailed Test

Journal: Experimental cell research

Article Title: VANGL2 protein stability is regulated by integrin αv and the extracellular matrix.

doi: 10.1016/j.yexcr.2018.11.017

Figure Lengend Snippet: Fig. 2. GFP-VANGL2 co-localizes with integrin αv at the plasma membrane. (A) GFP-VANGL2 transfected HT-1080 cells immunolabeled using an integrin αv (ITGAV) antibody show co-labeling at the plasma membrane (white arrows). (B) GFP-VANGL2 transfected cells immunolabeled using a paxillin antibody. At focal plane 1, paxillin-positive focal adhesions were observed that lack GFP-VANGL2 expression (white arrows). At focal plane 2, GFP-VANGL2 co-localizes with paxillin at certain plasma membrane domains (white arrows). Scale bars = 5 µm.

Article Snippet: The primary antibodies were VANGL2 (1:1000, MABN750), Millipore Sigma, St. Louis, MO; integrin αv (1:1000, ab17975), integrin β1 (1:1000, ab30394), and pan-cadherin (1:1000, ab16505) Abcam, Cambridge, MA; integrin α5 (1:1000, #4705), integrin β5 (1:1000, #3629), integrin β3 (1:1000, #13166), and β-actin (1:1000, #4967), Cell Signaling Technology, Danvers, MA; GAPDHHRP (1:2000, GTX627408-01), GeneTex, Inc., Irvine, CA.

Techniques: Clinical Proteomics, Membrane, Transfection, Immunolabeling, Labeling, Expressing

Journal: Experimental cell research

Article Title: VANGL2 protein stability is regulated by integrin αv and the extracellular matrix.

doi: 10.1016/j.yexcr.2018.11.017

Figure Lengend Snippet: Fig. 3. Integrin αv knockdown reduces Vangl2 protein in zebrafish. (A) Western blot of total protein from 24 h post-fertilization embryos. Protein was extracted from non-injected con- trol (NIC) and integrin αv (itgav) morpholino (MO) injected wild-type embryos. (B) Western blot of total protein isolated from non-injected and standard control morpholino injected wild-type embryos. (A, B) Box plots show median value ± standard deviation (whiskers) and the interquartile range (boxes) of likely variation (experiment performed three times, n = 13 biological replicates). Injection of in- tegrin αv morpholino caused a 20% average reduction in Vangl2 protein levels. *** P < 0.001; P value is versus NIC; two- tailed unpaired t-test.

Article Snippet: The primary antibodies were VANGL2 (1:1000, MABN750), Millipore Sigma, St. Louis, MO; integrin αv (1:1000, ab17975), integrin β1 (1:1000, ab30394), and pan-cadherin (1:1000, ab16505) Abcam, Cambridge, MA; integrin α5 (1:1000, #4705), integrin β5 (1:1000, #3629), integrin β3 (1:1000, #13166), and β-actin (1:1000, #4967), Cell Signaling Technology, Danvers, MA; GAPDHHRP (1:2000, GTX627408-01), GeneTex, Inc., Irvine, CA.

Techniques: Knockdown, Western Blot, Injection, Isolation, Control, Standard Deviation, Two Tailed Test

Journal: Experimental cell research

Article Title: VANGL2 protein stability is regulated by integrin αv and the extracellular matrix.

doi: 10.1016/j.yexcr.2018.11.017

Figure Lengend Snippet: Fig. 4. Opposing effects of integrin β3 and in- tegrin β5 knockdown on VANGL2 protein le- vels. (A) Western blots of total protein from non-targeting (NT) control and integrin β3 (ITGB3) knockdown HT-1080 cells. Integrin β3 protein is reduced by 89%. (B) Box plot shows median value ± standard deviation (whis- kers) and the interquartile range (boxes) of likely variation. Integrin β3 knockdown cells have increased VANGL2 protein levels (ex- periment performed three times, n = 9 biolo- gical replicates). (C) Western blots of total protein from NT control and integrin β5 knockdown cells. Integrin β5 protein is re- duced by 100%. (D) Box plot showing integrin β5 knockdown cells have decreased VANGL2 protein levels (experiment performed three times, n = 8 biological replicates). * P < 0.05; **P < 0.01; P values are versus NT siRNA; two-tailed unpaired t-test.

Article Snippet: The primary antibodies were VANGL2 (1:1000, MABN750), Millipore Sigma, St. Louis, MO; integrin αv (1:1000, ab17975), integrin β1 (1:1000, ab30394), and pan-cadherin (1:1000, ab16505) Abcam, Cambridge, MA; integrin α5 (1:1000, #4705), integrin β5 (1:1000, #3629), integrin β3 (1:1000, #13166), and β-actin (1:1000, #4967), Cell Signaling Technology, Danvers, MA; GAPDHHRP (1:2000, GTX627408-01), GeneTex, Inc., Irvine, CA.

Techniques: Knockdown, Western Blot, Control, Standard Deviation, Two Tailed Test

Journal: Experimental cell research

Article Title: VANGL2 protein stability is regulated by integrin αv and the extracellular matrix.

doi: 10.1016/j.yexcr.2018.11.017

Figure Lengend Snippet: Fig. 5. VANGL2 protein degradation is regu- lated by integrin αv. (A) Western blot of total protein from HT-1080 cells that were un- treated or treated with cycloheximide (cyc) as indicated (experiment performed three times, n = 3 biological replicates). Densitometry numbers for VANGL2 are normalized to actin. (B) Box plot shows the ratio of VANGL2 pro- tein in cyc treated/untreated non-targeting (NT) control and integrin αv (ITGAV) siRNA transfected HT-1080 cells (experiment per- formed three times, n = 11 biological re- plicates). The median value ± standard de- viation (whiskers) and the interquartile range (boxes) of likely variation are shown. (C) Western blots of total protein from NT and integrin αv knockdown cells untreated and treated with cycloheximide, MG-132, or chloroquine (chlor). These data were used to generate the box plots shown in panels B and D. (D) Box plot showing reduced VANGL2 protein levels in integrin αv knockdown cells are rescued by both MG-132 and chloroquine treatment (experiment performed three times, n = 8 biological replicates). * P < 0.05; **P < 0.01; ****P < 0.0001; P values are versus NT siRNA in panel B and as indicated in panel D; two-tailed unpaired t-test.

Article Snippet: The primary antibodies were VANGL2 (1:1000, MABN750), Millipore Sigma, St. Louis, MO; integrin αv (1:1000, ab17975), integrin β1 (1:1000, ab30394), and pan-cadherin (1:1000, ab16505) Abcam, Cambridge, MA; integrin α5 (1:1000, #4705), integrin β5 (1:1000, #3629), integrin β3 (1:1000, #13166), and β-actin (1:1000, #4967), Cell Signaling Technology, Danvers, MA; GAPDHHRP (1:2000, GTX627408-01), GeneTex, Inc., Irvine, CA.

Techniques: Western Blot, Control, Transfection, Knockdown, Two Tailed Test

Journal: Experimental cell research

Article Title: VANGL2 protein stability is regulated by integrin αv and the extracellular matrix.

doi: 10.1016/j.yexcr.2018.11.017

Figure Lengend Snippet: Fig. 6. Lysosomal VANGL2 localization in integrin αv knockdown cells. (A) Non-targeting (NT) control siRNA transfected cells expressing GFP-VANGL2 and im- munolabeled for LAMP1. Nuclei are labeled with DAPI (blue). Arrows denote plasma membrane expression. (B, C) Integrin αv (ITGAV) siRNA transfected cells expressing GFP-VANGL2 and with LAMP1 and DAPI labeling. a′-c′ Enlarged images of white-boxed regions in panels A-C. Certain double-positive vesicles are highlighted with arrowheads. Scale bars = 5 µm.

Article Snippet: The primary antibodies were VANGL2 (1:1000, MABN750), Millipore Sigma, St. Louis, MO; integrin αv (1:1000, ab17975), integrin β1 (1:1000, ab30394), and pan-cadherin (1:1000, ab16505) Abcam, Cambridge, MA; integrin α5 (1:1000, #4705), integrin β5 (1:1000, #3629), integrin β3 (1:1000, #13166), and β-actin (1:1000, #4967), Cell Signaling Technology, Danvers, MA; GAPDHHRP (1:2000, GTX627408-01), GeneTex, Inc., Irvine, CA.

Techniques: Knockdown, Control, Transfection, Expressing, Labeling, Clinical Proteomics, Membrane

Journal: Experimental cell research

Article Title: VANGL2 protein stability is regulated by integrin αv and the extracellular matrix.

doi: 10.1016/j.yexcr.2018.11.017

Figure Lengend Snippet: Fig. 7. Integrin activation stabilizes VANGL2 protein levels. (A) Fibronectin (Fn) immunolabeling of HT-1080 cells untreated (UT) or treated with MnCl2 to stimulate integrin activation and fibronectin assembly. Nuclei are labeled with DAPI (blue). Scale bars = 20 µm. (B) Box plot of cell adhesion assay data showing effect of MnCl2 treatment on non-targeting (NT) control and VANGL2 siRNA transfected cells (experiment performed three times, n = 6 biological replicates). The median value ± standard deviation (whiskers) and the interquartile range (boxes) of likely variation are shown. (C) Western blots of VANGL2 expression in untreated and MnCl2 treated cells plated on fibronectin and vitronectin. (D, E) Box plots showing VANGL2 protein levels are not increased in MnCl2 treated cells (experiments performed three times, n = 12 biological replicates). (F) Western blot of VANGL2 expression in cells that were untreated or treated with MnCl2 and/or cycloheximide (cyc). (G) Box plot showing the ratio of VANGL2 protein in cycloheximide treated/untreated cells with and without MnCl2 (experiment performed 6 times, n = 6 biological replicates). * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; P values are as indicated in panel B and versus VANGL2 expression in untreated cells in panels D and G; two-tailed unpaired t-test.

Article Snippet: The primary antibodies were VANGL2 (1:1000, MABN750), Millipore Sigma, St. Louis, MO; integrin αv (1:1000, ab17975), integrin β1 (1:1000, ab30394), and pan-cadherin (1:1000, ab16505) Abcam, Cambridge, MA; integrin α5 (1:1000, #4705), integrin β5 (1:1000, #3629), integrin β3 (1:1000, #13166), and β-actin (1:1000, #4967), Cell Signaling Technology, Danvers, MA; GAPDHHRP (1:2000, GTX627408-01), GeneTex, Inc., Irvine, CA.

Techniques: Activation Assay, Immunolabeling, Labeling, Cell Adhesion Assay, Control, Transfection, Standard Deviation, Western Blot, Expressing, Two Tailed Test

Journal: Experimental cell research

Article Title: VANGL2 protein stability is regulated by integrin αv and the extracellular matrix.

doi: 10.1016/j.yexcr.2018.11.017

Figure Lengend Snippet: Fig. 8. Inhibition of cell-matrix adhesion reduces VANGL2 protein levels. (A) Box plot of HT-1080 vitronectin cell adhesion assay data showing effect and titration of RGD peptide treatment. The median value ± standard deviation (whiskers) and the interquartile range (boxes) of likely variation are shown. (B) Western blots of untreated (UT) and RGD peptide treated cells plated on vi- tronectin (Vn) and fibronectin (Fn). (C) Box plot showing VANGL2 protein le- vels are decreased when integrin-vitronectin adhesion is inhibited (experiment performed four times, n = 10 biological replicates). ** P < 0.01; **** P < 0.0001; P values are versus untreated cells; two-tailed unpaired t-test.

Article Snippet: The primary antibodies were VANGL2 (1:1000, MABN750), Millipore Sigma, St. Louis, MO; integrin αv (1:1000, ab17975), integrin β1 (1:1000, ab30394), and pan-cadherin (1:1000, ab16505) Abcam, Cambridge, MA; integrin α5 (1:1000, #4705), integrin β5 (1:1000, #3629), integrin β3 (1:1000, #13166), and β-actin (1:1000, #4967), Cell Signaling Technology, Danvers, MA; GAPDHHRP (1:2000, GTX627408-01), GeneTex, Inc., Irvine, CA.

Techniques: Inhibition, Cell Adhesion Assay, Titration, Standard Deviation, Western Blot, Two Tailed Test

Journal: Experimental cell research

Article Title: VANGL2 interacts with integrin αv to regulate matrix metalloproteinase activity and cell adhesion to the extracellular matrix

doi: 10.1016/j.yexcr.2017.10.026

Figure Lengend Snippet: Src and paxillin activation in VANGL2 knockdown HT-1080 cells. Western blot analysis of Src and paxillin activation status in non-targeting control (NT) and VANGL2 (V2) siRNA transfected cells. Experiments were repeated five times and representative blots are shown. (A) P-Y416 (activated Src), Y416 negative control (non-phosphorylated), P-Y527 (inhibited Src), Y527 negative control (non-phosphorylated). (B) Quantification of panel (A) data. (C) P-Y118 (activated paxillin), total paxillin control. (D) Quantification of panel (C) data.

Article Snippet: Our western blot primary antibodies were [MMP14 (1:1000, MAB3328), EMD Millipore; VANGL2 (1:1000, MABN750), EMD Millipore; integrin αv (1:1000, ab17975), Abcam, Cambridge, MA), integrin β1 (1:1000, ab30394), Abcam; integrin α5 (1:1000, #4705), Cell Signaling Technology, Danvers, MA); integrin β3 (1:1000, #13166), Cell Signaling Technology; Src Antibody Sampler Kit (1:1000, 2107 non-phospho Src Y527; 1:1000, 2102 non-phospho Src Y416; 1:1000, 2105 phospho-Src Y527; 6943 phospho-Src Y416, Cell Signaling Technology); paxillin (1:1000, ab32084), Abcam; phospho-paxillin Y118 (1:1000, #2541), Cell Signaling Technology; GFP (1:1000, {"type":"entrez-nucleotide","attrs":{"text":"A10262","term_id":"490678","term_text":"A10262"}} A10262 ), Thermo Fisher Scientific; and GAPDH-HRP (1:2000, GTX627408-01), GeneTex, Inc., Irvine, CA].

Techniques: Activation Assay, Knockdown, Western Blot, Control, Transfection, Negative Control

Journal: Frontiers in Cell and Developmental Biology

Article Title: Hormone-Responsive BMP Signaling Expands Myoepithelial Cell Lineages and Prevents Alveolar Precocity in Mammary Gland

doi: 10.3389/fcell.2021.691050

Figure Lengend Snippet: BMPR1a deficiency resulted in precocious alveolar differentiation during pregnancy. (A) Immunohistochemistry staining for WAP in control and cKO mammary glands at pregnancy day 14.5. n = 3 mice per group. Scale bar, 50 μm. (B) Immunohistochemistry staining and western blotting for β-Casein in control and cKO mammary glands at pregnancy day 14.5. n = 3 mice per group. β-Tubulin was used as a loading control. Scale bar, 50 μm. Statistical analysis the expression of β-Casein/β-Tubulin. n = 3 mice. (C) qRT-PCR analysis of Wap , Lalba and Csn2 in mammary epithelial cells isolated from control and cKO mice at pregnancy day 14.5. n = 4–6 biological replicates. (D) Immunofluorescence staining and western blotting for Plin2 in control and cKO mammary glands at pregnancy day 14.5. n = 3 mice per group. GAPDH was used as a loading control. Scale bar, 25 μm. Statistical analysis the expression of Plin2/GAPDH. n = 3 mice. (E) Heatmap for milk protein-related gene expression in control and cKO mammary glands at pregnancy day 14.5. n = 4 mice per group. (F) Immunohistochemistry staining and western blotting for Stat5 and pStat5 in control and cKO mammary glands at pregnancy day 14.5. n = 3 mice per group. β-Actin was used as a loading control. Scale bar, 50 μm. Statistical analysis the expression of pStat5/β-Actin. n = 3 mice. (G) Immunohistochemistry staining for Prlr in control and cKO mammary glands at pregnancy day 14.5. n = 3 mice per group. Scale bar, 50 μm. (H) qRT-PCR analysis of Elf5, Tnfsf11, Src, Socs-1 , and Socs-2 in mammary epithelial cells isolated from control and cKO mice at pregnancy day 14.5. n ≥ 3 biological replicates. Data were presented as means ± SD. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: The following primary antibodies were used: BMPR1a (1:250, 12702-1-AP, Proteintech, Rosemont, United States), Sp1 (1:500, 21962-1-AP, Proteintech), K14 (1:400, ab7800, Abcam), K8 (1:800, ab59400, Abcam), pSmad1/5 (1:800, 9516, CST, Danvers, MA, United States), WAP (1:400, sc-374648, Santa Cruz, CA, United States), β-Casein (1:400, sc-166530, Santa Cruz), Plin2 (1:500, ab108323, Abcam), Stat5 (1:200, ab68465, Abcam), pStat5 (1:400, 9359, CST), Prlr (1:100, ab170935, Abcam), Ki67 (1:800, ab15580, Abcam), Cyclin D1 (1:1000, 55506, CST), Cleaved caspase3 (1:1000, 9664, CST), p63 (1:1000, ab124762, Abcam), Slug (1:400, 9585, CST), Smad4 (1:800, 46535, CST), P-cadherin (1:200, AF761, R&D, Minneapolis, MN, United States), GFP (1:1000, ab290, Abcam), BMP4 (1:200, MAB5020, R&D) and Integrin β3 (1:250, 13166, CST).

Techniques: Immunohistochemistry, Staining, Western Blot, Expressing, Quantitative RT-PCR, Isolation, Immunofluorescence