Journal: Respiratory Research

Article Title: Effect of active vitamin D3 on VEGF-induced ADAM33 expression and proliferation in human airway smooth muscle cells: implications for asthma treatment

doi: 10.1186/s12931-016-0490-9

Figure Lengend Snippet: 1,25(OH)2D3 inhibits VEGF-induced ADAM33 expression at both mRNA and protein level. ASM cells were incubated with various doses of 1,25(OH)2D3 for 9 h before treatment or not with 50 ng/ml of VEGF for 30 min, and then real-time PCR performed ( a ). ASM cells were incubated at indicated times of 100 nM of 1,25(OH)2D3, and then real-time PCR performed ( b ). The values are normalized relative to the GAPDH standard. ASM cells were incubated with various doses of 1,25(OH)2D3 for 24 h before treatment or not with 50 ng/ml of VEGF for 30 min, and then western blotting analysis for ADAM33 was performed ( c ). ASM cells were incubated at indicated times of 100 nM of 1,25(OH)2D3 before treatment or not with 50 ng/ml of VEGF for 30 min, and then western blotting analysis for ADAM33 was performed ( d ). β-actin was used as a loading control. All data are representative of three independent experiments. Values represent the means ± SEM. * P < 0.05 vs. control, # P < 0.05, ## P < 0.005 vs. VEGF alone

Article Snippet: ADAM33 siRNAs were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Expressing, Incubation, Real-time Polymerase Chain Reaction, Western Blot, Control

Journal: Respiratory Research

Article Title: Effect of active vitamin D3 on VEGF-induced ADAM33 expression and proliferation in human airway smooth muscle cells: implications for asthma treatment

doi: 10.1186/s12931-016-0490-9

Figure Lengend Snippet: 1,25(OH)2D3 inhibits cell proliferation by down-regulation of ADAM33 expression. ASM cells were incubated with various doses of 1,25(OH)2D3 for 48 h before treatment or not with 50 ng/ml of VEGF for 30 min, and then cell proliferation was determined by BrdU incorporation ( a ). ASM cells were incubated at indicated times of 100 nM of 1,25(OH)2D3 before treatment or not with 50 ng/ml of VEGF for 30 min, and then cell proliferation was determined by BrdU incorporation ( b ). ASM cells were transfected with negative siRNA or ADAM33 siRNA, and then real-time PCR performed. The values are normalized relative to the GAPDH standard ( c ). ASM cells ( d ) and ASM cells-ADAM33 ( e ) were transfected with negative siRNA or ADAM33 siRNA, and then western blotting analysis for ADAM33 was performed. β-actin was used as a loading control. ASM cells-ADAM33 were transfected with negative siRNA or ADAM33 siRNA in the presence of VEGF (50 ng/ml) and 1,25-(OH)2D3 (100 nM) for 48 h, and then cell proliferation was determined by BrdU incorporation ( f ). All experiments were done at least three times. Values represent the means ± SEM. * P < 0.05 vs. control or ASMs-vector; # P < 0.05 vs. VEGF alone or control siRNA or ASMs-control siRNA

Article Snippet: ADAM33 siRNAs were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Expressing, Incubation, BrdU Incorporation Assay, Transfection, Real-time Polymerase Chain Reaction, Western Blot, Control, Plasmid Preparation

Journal: Respiratory Research

Article Title: Effect of active vitamin D3 on VEGF-induced ADAM33 expression and proliferation in human airway smooth muscle cells: implications for asthma treatment

doi: 10.1186/s12931-016-0490-9

Figure Lengend Snippet: 1,25(OH)2D3 inhibits VEGF-induced ADAM33 expression and cell proliferation by inactivation of VEGFR2. ASM cells were incubated with indicated doses of SU1498 for 2 h before treatment with VEGF (50 ng/ml) for 24 h, and then western blotting analysis for ADAM33 was performed. β-actin was used as a loading control ( a ). ASM cells were incubated with indicated doses of SU1498 for 2 h before treatment with VEGF (50 ng/ml) for 48 h, and then cell proliferation was determined by BrdU incorporation ( b ). ASM cells were incubated with various doses of 1,25(OH)2D3 for 24 h before treatment or not with 50 ng/ml of VEGF for 30 min, and then western blotting analysis for VEGFR2 was performed ( c ). All experiments were done at least three times. Values represent the means ± SEM. * P < 0.05 vs. control; # P < 0.05, ## P < 0.005 vs. VEGF alone

Article Snippet: ADAM33 siRNAs were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Expressing, Incubation, Western Blot, Control, BrdU Incorporation Assay

Journal: Respiratory Research

Article Title: Effect of active vitamin D3 on VEGF-induced ADAM33 expression and proliferation in human airway smooth muscle cells: implications for asthma treatment

doi: 10.1186/s12931-016-0490-9

Figure Lengend Snippet: 1,25(OH)2D3 inhibits VEGF-induced ERK 1/2 phosphorylation in ASM cells. ASM cells were incubated at indicated times of VEGF (50 ng/ml), and then western blotting analysis for phospho-ERK 1/2 ( a ) and phospho-Akt ( b ) was performed. ASM cells were incubated with various doses of 1,25(OH)2D3 for 24 h before treatment or not with 50 ng/ml of VEGF for 30 min, and then western blotting analysis for phospho-ERK 1/2 ( c ) and phospho-Akt ( d ) was performed. The total ERK1/2 and Akt was used as a loading control. ASM cells were incubated with 20 μM U0126 or 20 μM LY294002 for 2 h before treatment with VEGF (50 ng/ml) for 24 h, and then western blotting analysis for ADAM33 was performed. β-actin was used as a loading control ( e ). ASM cells were incubated with 20 μM U0126 or 20 μM LY294002 for 2 h before treatment with VEGF (50 ng/ml) for 48 h, and then cell proliferation was determined by BrdU incorporation ( f ). All experiments were done at least three times. Values represent the means ± SEM. * P < 0.05 vs. control; # P < 0.05 vs. VEGF alone

Article Snippet: ADAM33 siRNAs were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Phospho-proteomics, Incubation, Western Blot, Control, BrdU Incorporation Assay