mouse aorta vsmc  (Keygen Biotech)

Journal: The Journal of Biological Chemistry

Article Title: Angiogenic factor AGGF1 blocks neointimal formation after vascular injury via interaction with integrin α7 on vascular smooth muscle cells

doi: 10.1016/j.jbc.2022.101759

Figure Lengend Snippet: Wild-type and mutant AGGF1-C1 and C2, but not AGGF1-C3, increase the expression of VSMC phenotypic switching markers α-SMA, SM22, and MYH11 in VSMCs. A , Western blot analysis for α-SMA, SM22, and MYH11. MOVAS cells were treated with 20 μl control PBS or 20 μl of 5 μg/ml wild-type AGGF1 (AGGF1-WT) or mutant AGGF1 (AGGF1-C1, AGGF1-C2 and AGGF1-C3) for 24 h, lysed, and used for Western blot analysis. B , quantification of Western blot images as in ( A ) (mean ± SD, one-way ANOVA with Dunnett test for multiple comparison; ∗ p < 0.05, ∗∗ p < 0.01, n = 3/group). C , luciferase assays showing that AGGF1-WT, AGGF1-C1 and AGGF1-C2 increased transcriptional activation of VSMC contractile marker genes encoding MYH11, a-SMA and SM22 in the presence of SRF (serum response factor), but the effects were not observed for AGGF1-C3. MOVAS cells were cotransfected with an expression plasmid for SRF together with a MYH11 , ACTA2 , or TAGLN promoter luciferase reporter gene with or without an expression plasmid for wild type or mutant AGGF1 . Cells were lysed and used for luciferase assays 48 h after transfection. NC, empty vector. Data are shown as mean ± SD (one-way ANOVA with Dunnett test for multiple comparison; ∗ p < 0.05, n = 3/group). NS, not significant. AGGF1, angiogenic factor with G patch and FHA domains 1; VSMCs, vascular smooth muscle cells.

Article Snippet: MOVAS-1, an immortalized mouse aorta VSMC line, was purchased from ATCC (American Type Culture Collection).

Techniques: Mutagenesis, Expressing, Western Blot, Control, Comparison, Luciferase, Activation Assay, Marker, Plasmid Preparation, Transfection

Journal: The Journal of Biological Chemistry

Article Title: Angiogenic factor AGGF1 blocks neointimal formation after vascular injury via interaction with integrin α7 on vascular smooth muscle cells

doi: 10.1016/j.jbc.2022.101759

Figure Lengend Snippet: Mutant AGGF1-C3 lost the effect of AGGF1 on cell proliferation, cell cycle regulation, and migration of vascular smooth muscle cells. A and B , MOVAS cells were incubated with 20 μl of wild-type AGGF1 (AGGF1-WT) (5 μg/ml) or mutant AGGF1 AGGF1-C1, AGGF1-C2, and AGGF1-C3 (5 μg/ml) versus 20 μl PBS control for 36 h ( A ) or 24 h ( B – D ), and used for cell proliferation assays ( A ) and cell cycle analysis ( B ). AGGF1-WT, AGGF1-C1, and AGGF1-C2 inhibited the proliferation of VSMCs, but this effect was not observed for AGGF1-C3. A , cell proliferation assays were performed with the CCK8 kit. Data are shown as mean ± SD (one-way ANOVA with Dunnett test for multiple comparison; ∗ p < 0.05, n = 3/group). AGGF1-WT, AGGF1-C1 and AGGF1-C2, but not AGGF1-C3, significantly decreased the number of S-phase cells ( B ). Cell cycle analysis was performed and the number of S-phase cells was measured by flow cytometry. Data are shown as mean ± SD (one-way ANOVA with Dunnett test for multiple comparison; ∗ p < 0.05, n = 3/group). C , AGGF1-WT, AGGF1-C1 and AGGF1-C2, but not AGGF1-C3, inhibited the VSMC migration in scratch-wound healing assays. MOVAS cells were cultured in a 6-well plate overnight, and a scratch was made on the bottom of wells. A total of 20 μl of wild type AGGF1 protein (AGGF1-WT) (5 μg/ml), mutant AGGF1 proteins AGGF1-C1, AGGF1-C2 and AGGF1-C3 (5 μg/ml) or PBS control was added. The cells were allowed to migrate for 24 h. The degree of cell migration was quantified and shown on the right. D , AGGF1-WT, AGGF1-C1, and AGGF1-C2, but not AGGF1-C3, inhibited expression of cyclin D and upregulated p27 and p21. MOVAS cells were treated as above, lysed, and used for Western blot analysis. E , quantification of Western blot images as in ( D ) (mean ± SD, one-way ANOVA with Dunnett test for multiple comparison; ∗∗ p < 0.01, n = 3/group). NS, not significant. AGGF1, angiogenic factor with G patch and FHA domains 1; VSMCs, vascular smooth muscle cells.

Article Snippet: MOVAS-1, an immortalized mouse aorta VSMC line, was purchased from ATCC (American Type Culture Collection).

Techniques: Mutagenesis, Migration, Incubation, Control, Cell Cycle Assay, Comparison, Flow Cytometry, Cell Culture, Expressing, Western Blot