rhdll4  (R&D Systems)

Journal: Angiogenesis

Article Title: RHOQ is induced by DLL4 and regulates angiogenesis by determining the intracellular route of the Notch intracellular domain

doi: 10.1007/s10456-020-09726-w

Figure Lengend Snippet: RHOQ is induced by DLL4/Notch signalling in vitro. HUVECs were grown on BSA or rhDLL4-coated plates, harvested at indicated time points and changes in RHOQ and DLL4/Notch targets were assessed by a QPCR or b by western blotting. c Active RHOQ status following GTPase pull down assay and magnitude of active RHOQ was detected by western blotting. Changes in DLL4/Notch target expression following 16 h incubation with DBZ (20 nM) compared to DMSO equivalent controls was assessed by either. d QPCR, normalised to BSA DMSO control, or e by western blotting, or f cells were fixed and analysed for NICD bound to Notch promoter binding site for RHOQ, compared to that of DLL4 and HEY1 by chromatin immunoprecipitation. β-Actin as a loading control and densitometry was performed on western blots. Densitometry ratios were expressed relative to the first BSA control sample (Error bars = S.D. Key: * p < 0.05, ** p < 0.01, *** p < 0.0001 one-way ANOVA or unpaired Student’s t -test comparing two data groups; data representative of n = 3 independent experiments)

Article Snippet: HUVECs were seeded onto dishes pre-coated with BSA (1 μg/ml; Sigma-Aldrich) or rhDLL4 extracellular domain (1 μg/ml; R&D Systems, Minneapolis, USA) in 0.2% (w/v) gelatin (Sigma-Aldrich).

Techniques: In Vitro, Western Blot, Pull Down Assay, Expressing, Incubation, Binding Assay, Chromatin Immunoprecipitation

Journal: Angiogenesis

Article Title: RHOQ is induced by DLL4 and regulates angiogenesis by determining the intracellular route of the Notch intracellular domain

doi: 10.1007/s10456-020-09726-w

Figure Lengend Snippet: Loss of RHOQ expression abolished expression of downstream DLL4/Notch signalling. HUVECs were either transfected with siRHOQ or infected with human RHOQ (hRHOQ); cells were then grown on BSA or rhDLL4-coated plates and harvested at indicated time points. Changes in DLL4/Notch downstream target expression in stimulated siRHOQ transfected HUVECs were assessed by a QPCR after 16 h stimulation, expressed relative to BSA control, and b western blotting, after either (i) 8 h or (ii) 24 h stimulation, using B-Actin as a loading control. c Changes in DLL4/Notch downstream target expression in stimulated HUVECs overexpressing hRHOQ was assessed over time by QPCR. Densitometry ratios were expressed relative to the first BSA control sample (Error bars = S.D. Key: * p < 0.05, ** p < 0.01, *** p < 0.0001 one-way ANOVA; data representative of n = 3 independent experiments)

Article Snippet: HUVECs were seeded onto dishes pre-coated with BSA (1 μg/ml; Sigma-Aldrich) or rhDLL4 extracellular domain (1 μg/ml; R&D Systems, Minneapolis, USA) in 0.2% (w/v) gelatin (Sigma-Aldrich).

Techniques: Expressing, Transfection, Infection, Western Blot

Journal: Angiogenesis

Article Title: RHOQ is induced by DLL4 and regulates angiogenesis by determining the intracellular route of the Notch intracellular domain

doi: 10.1007/s10456-020-09726-w

Figure Lengend Snippet: Localisation changes of NICD in RHOQ negative cells. a Transfected HUVECs with RHOQ siRNA duplexes were cultured on BSA or rhDLL4 (1 μg/ml)-coated plates for 8 h for NICD analysis or 16 h for other markers before immuno-fluorescence staining for a RHOQ (green) and Exo70 (red), b NICD (green) with RHOQ (red) or Exo70 (red) and visualised by confocal microscopy. Nuclei stained with DAPI (blue). (Key: Scale bar = 20 nm; data representative of n = 3 independent experiments)

Article Snippet: HUVECs were seeded onto dishes pre-coated with BSA (1 μg/ml; Sigma-Aldrich) or rhDLL4 extracellular domain (1 μg/ml; R&D Systems, Minneapolis, USA) in 0.2% (w/v) gelatin (Sigma-Aldrich).

Techniques: Transfection, Cell Culture, Fluorescence, Staining, Confocal Microscopy

Journal: Angiogenesis

Article Title: RHOQ is induced by DLL4 and regulates angiogenesis by determining the intracellular route of the Notch intracellular domain

doi: 10.1007/s10456-020-09726-w

Figure Lengend Snippet: RHOQ/Exo70 co-localise with Notch components. a HUVECs were transfected with lentivirus to overexpress GFP tagged RHOQ and cultured on BSA or rhDLL4-coated plates and fixed 24 h later and Duo-link staining detecting only Exo70/NICD (red) associated antibodies, visualised by confocal microscopy. Nuclei stained with DAPI (blue). b Immunoprecipitation with either IgG control antibody, RHOQ antibody and Exo70 antibody and expression of Notch components assessed by western blotting, compared to whole cell (W) lysates, with B-Actin used as a loading control. (Key: White arrow, example of co-localisation; Scale bar = 20 nm; data representative of n = 3 independent experiments)

Article Snippet: HUVECs were seeded onto dishes pre-coated with BSA (1 μg/ml; Sigma-Aldrich) or rhDLL4 extracellular domain (1 μg/ml; R&D Systems, Minneapolis, USA) in 0.2% (w/v) gelatin (Sigma-Aldrich).

Techniques: Transfection, Cell Culture, Staining, Confocal Microscopy, Immunoprecipitation, Expressing, Western Blot

Journal: Angiogenesis

Article Title: RHOQ is induced by DLL4 and regulates angiogenesis by determining the intracellular route of the Notch intracellular domain

doi: 10.1007/s10456-020-09726-w

Figure Lengend Snippet: NICD does not translocate to the nucleus in RHOQ negative cells. a Transfected HUVECs with RHOQ siRNA duplexes were cultured on BSA or rhDLL4 (1 μg/ml)-coated plates for 8 h before ( a ) cells were pelleted and lysed for whole cell lysate (W), or cytoplasmic fraction (C), nuclear fraction (N) and changes in the NICD localisation assessed by western blotting, using B-Actin and Lamin B as markers of fraction contamination and loading control or being fixed and b analysed for NICD binding to the Notch promoter binding site for DLL4 and Hey1 by chromatin immunoprecipitation (Data representative of n = 3 independent experiments)

Article Snippet: HUVECs were seeded onto dishes pre-coated with BSA (1 μg/ml; Sigma-Aldrich) or rhDLL4 extracellular domain (1 μg/ml; R&D Systems, Minneapolis, USA) in 0.2% (w/v) gelatin (Sigma-Aldrich).

Techniques: Transfection, Cell Culture, Western Blot, Binding Assay, Chromatin Immunoprecipitation

Journal: Angiogenesis

Article Title: RHOQ is induced by DLL4 and regulates angiogenesis by determining the intracellular route of the Notch intracellular domain

doi: 10.1007/s10456-020-09726-w

Figure Lengend Snippet: Loss of RHOQ increases autophagy and leads to degradation of Notch1 receptor. HUVECs cultured on BSA or rhDLL4 (1 μg/ml)-coated plates and effects on autophagosomes were assessed 16 h later by staining cells with autophagy tracker (red) with changes in tracker levels, a visualised by confocal microscopy (nuclei stained with DAPI (blue)), and assessed by b FACs or c cells were fixed and immuno-fluorescence staining for localisation changes in RHOQ (red) or Notch1 (red) and LC3B (green) proteins and visualised by confocal microscopy (nuclei stained with DAPI (blue)) or d illustrating LC3B binding sites on Notch1 (Error bars = S.D. Key: * p < 0.05, ** p < 0.01, *** p < 0.0001 one-way ANOVA or unpaired Student’s t -test between data group and control, Scale bar = 20 nm; representative images and data of n = 3 independent experiments)

Article Snippet: HUVECs were seeded onto dishes pre-coated with BSA (1 μg/ml; Sigma-Aldrich) or rhDLL4 extracellular domain (1 μg/ml; R&D Systems, Minneapolis, USA) in 0.2% (w/v) gelatin (Sigma-Aldrich).

Techniques: Cell Culture, Staining, Confocal Microscopy, Fluorescence, Binding Assay

Journal: Angiogenesis

Article Title: RHOQ is induced by DLL4 and regulates angiogenesis by determining the intracellular route of the Notch intracellular domain

doi: 10.1007/s10456-020-09726-w

Figure Lengend Snippet: Loss of RHOQ expression leads to NICD degradation in lysosomes. HUVECs cultured on BSA or rhDLL4 (1 μg/ml)-coated plates and effects on lysosomes were assessed 16 h later by staining cells with lysosome tracker (red) with changes in tracker levels, a visualised by confocal microscopy (nuclei stained with DAPI (blue)) and assessed by b FACs or cells were fixed and immuno-fluorescence staining for localisation changes in proteins c duo-link staining detecting only Lamp/Notch1 (red) or Lamp1/NICD (green) associated antibodies, visualised by confocal microscopy. Nuclei stained with DAPI (blue). d Transfected HUVECs with RHOQ siRNA duplexes were cultured on BSA or rhDLL4 (1 μg/ml)-coated plates for 8 h with or without chloroquine (10 µM) before ( a ) changes in DLL4/Notch downstream target expression by QPCR (Error bars = S.D. Key: * p < 0.05, ** p < 0.01, *** p < 0.0001 one-way ANOVA or unpaired Student’s t -test between data group and control, Scale bar = 20 nm; representative images and data of n = 3 independent experiments)

Article Snippet: HUVECs were seeded onto dishes pre-coated with BSA (1 μg/ml; Sigma-Aldrich) or rhDLL4 extracellular domain (1 μg/ml; R&D Systems, Minneapolis, USA) in 0.2% (w/v) gelatin (Sigma-Aldrich).

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