Journal: Autophagy

Article Title: Autophagy-induced KDR/VEGFR-2 activation promotes the formation of vasculogenic mimicry by glioma stem cells

doi: 10.1080/15548627.2017.1336277

Figure Lengend Snippet: Autophagy promotes tubule-like structure formation by GSCs in 3D scaffold. (A) GSCs transfected with EGFP-LC3 were cultured in DMEM, STM or EBM for 72 h. Confocal microscopy was used to assess autophagy shown as green spots (left panel). Two hundred cells were randomly chosen to count the number of EGFP-LC3 fluorescent spots (right panel). Scale bar: 10 μm. (B) Confocal microscopy showing EGFP-transfected GSCs to form tubule-like structure in 3D scaffold (left panel). The number of tubular structures formed by GSCs in 3D scaffold was analyzed (right pannel). Scale bar: 20 μm. Asterisks (*) indicate VM channel formed by tumor cells. (C) Expression of autophagy proteins, ATG5, ATG7, LC3-I to LC3-II conversion and SQSTM1. TUBB/β-Tubulin served as a control. (D) The expressions of PROM1, GFAP, KDR proteins and the phosphorylation of Y1175 in the C-terminal region of KDR (p-KDR) as well as CDH5 and LAMB2. (E) Double staining of LC3 and CDH5 as well as LC3 and LAMB2 in GSCs cultured with EBM on the scaffold. LC3 is marked as red spots in the cytoplasm; CDH5 and LAMB2 are in green color in the cytoplasm. Scale bar: 20 μm. Nuclei were stained by Hoechst 33324 in blue. The results are expressed as the mean ± SEM from 3 experiments.

Article Snippet: Antibodies Antibodies used include mouse anti-LAMB2/laminin B2 monoclonal antibody (mAb; BD Biosciences, 610722), rabbit anti-CDH5/cadherin 5 mAb (Cell Signaling Technology, 2158), rabbit anti-PROM1/CD133 polyclonal antibody (pAb; Santa Cruz Biotechnology, sc-30220), rabbit anti-GFAP pAb (Abcam, ab7260), mouse anti-CD34 mAb (Abcam, ab187282), rabbit anti-VEGF pAb (Proteintech, 19003–1-AP), rabbit anti-phospho- KDR/VEGFR-2 mAb (Cell Signaling Technology, 2478), rabbit anti-KDR/VEGFR-2 mAb (Cell Signaling Technology, 2479), mouse anti-LC3B mAb (Santa Cruz Biotechnology, sc-376404), rabbit anti-ATG5 mAb (Cell Signaling Technology, 8540), rabbit anti-ATG7 mAb (Cell Signaling Technology, 8558), rabbit anti-SQSTM1/p62 pAb (Sigma-Aldrich, P0067), rabbit anti-BECN1 mAb (Abcam, ab51031), mouse anti-PI3K p85 mAb (Abcam, ab86714), rabbit anti-pan-AKT pAb (Abcam, ab8805), rabbit anti-phospho-PI3K p85 (Y607) pAb (Abcam, ab182651), rabbit anti-phospho-pan-AKT pAb (Abcam, ab38449).

Techniques: Transfection, Cell Culture, Confocal Microscopy, Expressing, Double Staining, Staining

Journal: Autophagy

Article Title: Autophagy-induced KDR/VEGFR-2 activation promotes the formation of vasculogenic mimicry by glioma stem cells

doi: 10.1080/15548627.2017.1336277

Figure Lengend Snippet: Tubular structure formation by GSCs is promoted by rapamycin (RAPA). (A) RAPA (100 nM) was added to EBM and confocal microscopy was used to detect autophagic vesicles in GSCs forming tubular structure (left panel). Two hundred cells were randomly chosen to count green fluorescent spots within the cells (right panel). Scale bar: 10 μm. (B) Confocal microscopy showing EGFP-transfected GSCs to form tubule-like structure in 3D scaffold (left panel). The number of tubular structures was counted in the scaffold cultured with or without RAPA (right panel). Scale bar: 20 μm. Asterisks (*) indicate VM channel formed by tumor cells. (C) The expression of LC3-I to LC3-II conversion, SQSTM1 and VM-related proteins (KDR, p-KDR, CDH5 and LAMB2) in GSCs treated by RAPA. The results are expressed as the mean ± SEM from 3 experiments.

Article Snippet: Antibodies Antibodies used include mouse anti-LAMB2/laminin B2 monoclonal antibody (mAb; BD Biosciences, 610722), rabbit anti-CDH5/cadherin 5 mAb (Cell Signaling Technology, 2158), rabbit anti-PROM1/CD133 polyclonal antibody (pAb; Santa Cruz Biotechnology, sc-30220), rabbit anti-GFAP pAb (Abcam, ab7260), mouse anti-CD34 mAb (Abcam, ab187282), rabbit anti-VEGF pAb (Proteintech, 19003–1-AP), rabbit anti-phospho- KDR/VEGFR-2 mAb (Cell Signaling Technology, 2478), rabbit anti-KDR/VEGFR-2 mAb (Cell Signaling Technology, 2479), mouse anti-LC3B mAb (Santa Cruz Biotechnology, sc-376404), rabbit anti-ATG5 mAb (Cell Signaling Technology, 8540), rabbit anti-ATG7 mAb (Cell Signaling Technology, 8558), rabbit anti-SQSTM1/p62 pAb (Sigma-Aldrich, P0067), rabbit anti-BECN1 mAb (Abcam, ab51031), mouse anti-PI3K p85 mAb (Abcam, ab86714), rabbit anti-pan-AKT pAb (Abcam, ab8805), rabbit anti-phospho-PI3K p85 (Y607) pAb (Abcam, ab182651), rabbit anti-phospho-pan-AKT pAb (Abcam, ab38449).

Techniques: Confocal Microscopy, Transfection, Cell Culture, Expressing

Journal: Autophagy

Article Title: Autophagy-induced KDR/VEGFR-2 activation promotes the formation of vasculogenic mimicry by glioma stem cells

doi: 10.1080/15548627.2017.1336277

Figure Lengend Snippet: Autophagy mediates the formation of VM through KDR phosphorylation. (A) Confocal microscopy detection of autophagic vesicles formed by GSCs transfected with EGFP-LC3 and treated with 5 μM chloroquine (CQ) (left panel). Two hundred cells were randomly chosen to count green fluorescent spots within the cells (right panel). Scale bar: 10 μm. (B) Confocal microscopy showing EGFP-transfected GSCs to form vascular tubules in 3D scaffold (left panel). The number of tubular structures was counted in the scaffold with or without CQ (right panel). Scale bar: 20 μm. Asterisks (*) indicate VM channels formed by tumor cells. (C) The expression of LC3-I to LC3-II conversion, SQSTM1 and KDR, p-KDR, LAMB2, and CDH5 in GSCs after 5 μM CQ treatment. (D) Western blot detection of p-KDR, KDR, LAMB2, CDH5 and SQSTM1 in GSCs in medium with 100 μg/ml BEV and/or 5 μM CQ. The results are expressed as the mean ± SEM from 3 experiments.

Article Snippet: Antibodies Antibodies used include mouse anti-LAMB2/laminin B2 monoclonal antibody (mAb; BD Biosciences, 610722), rabbit anti-CDH5/cadherin 5 mAb (Cell Signaling Technology, 2158), rabbit anti-PROM1/CD133 polyclonal antibody (pAb; Santa Cruz Biotechnology, sc-30220), rabbit anti-GFAP pAb (Abcam, ab7260), mouse anti-CD34 mAb (Abcam, ab187282), rabbit anti-VEGF pAb (Proteintech, 19003–1-AP), rabbit anti-phospho- KDR/VEGFR-2 mAb (Cell Signaling Technology, 2478), rabbit anti-KDR/VEGFR-2 mAb (Cell Signaling Technology, 2479), mouse anti-LC3B mAb (Santa Cruz Biotechnology, sc-376404), rabbit anti-ATG5 mAb (Cell Signaling Technology, 8540), rabbit anti-ATG7 mAb (Cell Signaling Technology, 8558), rabbit anti-SQSTM1/p62 pAb (Sigma-Aldrich, P0067), rabbit anti-BECN1 mAb (Abcam, ab51031), mouse anti-PI3K p85 mAb (Abcam, ab86714), rabbit anti-pan-AKT pAb (Abcam, ab8805), rabbit anti-phospho-PI3K p85 (Y607) pAb (Abcam, ab182651), rabbit anti-phospho-pan-AKT pAb (Abcam, ab38449).

Techniques: Confocal Microscopy, Transfection, Expressing, Western Blot

Journal: Autophagy

Article Title: Autophagy-induced KDR/VEGFR-2 activation promotes the formation of vasculogenic mimicry by glioma stem cells

doi: 10.1080/15548627.2017.1336277

Figure Lengend Snippet: Knockdown of ATG5 reduces tubular structure formation by GSCs and phosphorylation of KDR. (A) The expression of ATG5, p-KDR, KDR, CDH5 and LAMB2 in GSCs transfected with siATG5. (B) Two hundred cells were randomly chosen to count green fluorescent spots within the cells. (C) The number of tubular structures formed by GSCs and siATG5-GSCs in the scaffold counted with confocal microscopy. (D) Western blot detection of p-KDR and KDR in siATG5-GSCs cultured with 100 μg/ml BEV. (E) and (F) Mice with intracranial injection of GSCs transfected with siCtrl RNA or siATG5 were treated with BEV. Forty-two animals were then randomly divided into 7 groups consisting of 6 animals each. After 4 wk, tumors were collected to analyze vessel density (E) and formation of VM (F). HPF, high-power field. The results are expressed as the mean ± SEM from 3 experiments.

Article Snippet: Antibodies Antibodies used include mouse anti-LAMB2/laminin B2 monoclonal antibody (mAb; BD Biosciences, 610722), rabbit anti-CDH5/cadherin 5 mAb (Cell Signaling Technology, 2158), rabbit anti-PROM1/CD133 polyclonal antibody (pAb; Santa Cruz Biotechnology, sc-30220), rabbit anti-GFAP pAb (Abcam, ab7260), mouse anti-CD34 mAb (Abcam, ab187282), rabbit anti-VEGF pAb (Proteintech, 19003–1-AP), rabbit anti-phospho- KDR/VEGFR-2 mAb (Cell Signaling Technology, 2478), rabbit anti-KDR/VEGFR-2 mAb (Cell Signaling Technology, 2479), mouse anti-LC3B mAb (Santa Cruz Biotechnology, sc-376404), rabbit anti-ATG5 mAb (Cell Signaling Technology, 8540), rabbit anti-ATG7 mAb (Cell Signaling Technology, 8558), rabbit anti-SQSTM1/p62 pAb (Sigma-Aldrich, P0067), rabbit anti-BECN1 mAb (Abcam, ab51031), mouse anti-PI3K p85 mAb (Abcam, ab86714), rabbit anti-pan-AKT pAb (Abcam, ab8805), rabbit anti-phospho-PI3K p85 (Y607) pAb (Abcam, ab182651), rabbit anti-phospho-pan-AKT pAb (Abcam, ab38449).

Techniques: Expressing, Transfection, Confocal Microscopy, Western Blot, Cell Culture, Injection

Journal: International Journal of Molecular Sciences

Article Title: The Role of Sensory Nerves in Dental Pulp Homeostasis: Histological Changes and Cellular Consequences after Sensory Denervation

doi: 10.3390/ijms25021126

Figure Lengend Snippet: Antibodies used in this study.

Article Snippet: Laminin , Rabbit , Boster , PA1581.

Techniques:

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Pathogenicity of a Human Laminin β 2 Mutation Revealed in Models of Alport Syndrome

doi: 10.1681/ASN.2017090997

Figure Lengend Snippet: LAMB2-S83R increases Alport-associated deposition of ectopic laminins into the GBM. Frozen sections of kidneys from 6- to 10-week-old mice of the indicated genotypes were stained for LAMB2 (6 weeks shown), LAMB1 (10 weeks shown), and LAMA2 (10 weeks shown for control and Lamb2+/+; Col4a3−/− and 8 weeks shown for Lamb2+/S83R; Col4a3−/−). LAMB2 was detected in the GBM in all cases. LAMB1 and LAMA2 are normally in the mesangial matrix but were detected ectopically in the Alport GBM regardless of Lamb2 genotype, although the levels appeared slightly increased in some Lamb2+/S80R; Col4a3−/− versus Lamb2+/+; Col4a3−/− glomeruli. Colocalization with GBM agrin is shown in Supplemental Figures 3 and 4; n=4–5.

Article Snippet: For immunofluorescence, kidneys were frozen in OCT and cryosectioned at 7 μ m. Antibodies, including rabbit anti-mouse LAMB1 and LAMB2, 52 mouse anti-rat LAMB2, 24 rat anti-mouse LAMA2 (clone 4H8–2; Axxora), 53 rat anti-mouse nidogen (Clone ELM1), mouse anti-bovine COL4A345, 54 and rabbit anti-mouse agrin, 55 were diluted into PBS containing 1% BSA.

Techniques: Staining

Journal: Aging (Albany NY)

Article Title: MicroRNA-29a-3p delivery via exosomes derived from engineered human mesenchymal stem cells exerts tumour suppressive effects by inhibiting migration and vasculogenic mimicry in glioma

doi: 10.18632/aging.202424

Figure Lengend Snippet: miR-29a-3p inhibited migration and VM formation in glioma cells. ( A ) The effect of miR-29a-3p on cell movement was assessed using transwell migration assays (scale bar, 100 μm; n=3). ( B ) Quantification of transwell migration assays in ( A ). Data are shown as the mean±SD, n=3, one-way ANOVA (*, P < 0.05). ( C ) Effect of miR-29a-3p on VM formation ability (scale bar, 200 μm; n=3). ( D ) Quantification of relative VM number in ( C ). Data are shown as the mean±SD, n=3, one-way ANOVA (*, P < 0.05). ( E ) Western blot analysis of protein levels of N-cadherin, MMP9 and LAMB2 in A172 and U87 cells. GAPDH was used as a whole-cell protein loading control. Results are from three independent experiments.

Article Snippet: The blots were incubated with primary antibodies against ROBO1 (rabbit anti-ROBO1 polyclonal antibody, 1:500 dilution, 20219-1-AP; Proteintech; China), LAMB2 (rabbit anti-LAMB2 polyclonal antibody, 1:1000 dilution, 10895-1-AP; Proteintech; China), N-cadherin, MMP9, and GAPDH (1:1000; Cell Signaling Technology; USA).

Techniques: Migration, Western Blot, Control

Journal: Aging (Albany NY)

Article Title: MicroRNA-29a-3p delivery via exosomes derived from engineered human mesenchymal stem cells exerts tumour suppressive effects by inhibiting migration and vasculogenic mimicry in glioma

doi: 10.18632/aging.202424

Figure Lengend Snippet: miR-29a-3p inhibited migration and VM formation by directly targeting ROBO1. ( A ) Schematic representation of the predicted binding sites for miR-29a-3p in the ROBO1 3′-UTR (wild type; WT) and the designed mutant versions (mutant; MUT) of the ROBO1 3’-UTR (left panel). Relative luciferase activity of HEK293T cells in the presence of the indicated treatments (middle and right plots). Data are shown as the mean±SD, n=3, one-way ANOVA (*, P < 0.05). ( B ) Western blot analysis of the protein level of ROBO1,N-cadherin, MMP9 and LAMB2 after miR-29a-3p transfection. Results are from three independent experiments. ( C ) Western blot analysis of the expression of ROBO1,N-cadherin, MMP9 and LAMB2 after ROBO1 and miR-29a-3p overexpression. Results are from three independent experiments. ( D ) Representative images for the transwell assay (scale bar, 100 μm; n=3). ( E ) Quantification of transwell migration assays in ( D ). Data are shown as the mean±SD, n=3, one-way ANOVA (*, P < 0.05). ( F ) Representative images for the VM formation assay (scale bar, 200 μm; n=3). ( G ) Quantification of relative VM numbers in ( F ). Data are shown as the mean±SD, n=3, one-way ANOVA (*, P < 0.05).

Article Snippet: The blots were incubated with primary antibodies against ROBO1 (rabbit anti-ROBO1 polyclonal antibody, 1:500 dilution, 20219-1-AP; Proteintech; China), LAMB2 (rabbit anti-LAMB2 polyclonal antibody, 1:1000 dilution, 10895-1-AP; Proteintech; China), N-cadherin, MMP9, and GAPDH (1:1000; Cell Signaling Technology; USA).

Techniques: Migration, Binding Assay, Mutagenesis, Luciferase, Activity Assay, Western Blot, Transfection, Expressing, Over Expression, Transwell Assay, Tube Formation Assay

Journal: Aging (Albany NY)

Article Title: MicroRNA-29a-3p delivery via exosomes derived from engineered human mesenchymal stem cells exerts tumour suppressive effects by inhibiting migration and vasculogenic mimicry in glioma

doi: 10.18632/aging.202424

Figure Lengend Snippet: miR-29a-3p inhibited migration and VM formation by directly targeting ROBO1. ( A ) Schematic representation of the predicted binding sites for miR-29a-3p in the ROBO1 3′-UTR (wild type; WT) and the designed mutant versions (mutant; MUT) of the ROBO1 3’-UTR (left panel). Relative luciferase activity of HEK293T cells in the presence of the indicated treatments (middle and right plots). Data are shown as the mean±SD, n=3, one-way ANOVA (*, P < 0.05). ( B ) Western blot analysis of the protein level of ROBO1,N-cadherin, MMP9 and LAMB2 after miR-29a-3p transfection. Results are from three independent experiments. ( C ) Western blot analysis of the expression of ROBO1,N-cadherin, MMP9 and LAMB2 after ROBO1 and miR-29a-3p overexpression. Results are from three independent experiments. ( D ) Representative images for the transwell assay (scale bar, 100 μm; n=3). ( E ) Quantification of transwell migration assays in ( D ). Data are shown as the mean±SD, n=3, one-way ANOVA (*, P < 0.05). ( F ) Representative images for the VM formation assay (scale bar, 200 μm; n=3). ( G ) Quantification of relative VM numbers in ( F ). Data are shown as the mean±SD, n=3, one-way ANOVA (*, P < 0.05).

Article Snippet: The blots were incubated with primary antibodies against ROBO1 (rabbit anti-ROBO1 polyclonal antibody, 1:500 dilution, 20219-1-AP; Proteintech; China), LAMB2 (rabbit anti-LAMB2 polyclonal antibody, 1:1000 dilution, 10895-1-AP; Proteintech; China), N-cadherin, MMP9, and GAPDH (1:1000; Cell Signaling Technology; USA).

Techniques: Migration, Binding Assay, Mutagenesis, Luciferase, Activity Assay, Western Blot, Transfection, Expressing, Over Expression, Transwell Assay, Tube Formation Assay