Figure 4
From: MicroRNA miR-509 Regulates ERK1/2, the Vimentin Network, and Focal Adhesions by Targeting Plk1
Vimentin network organization and focal adhesion size are regulated by miR-509. (A) HASM cells treated with miR-control or miR-509, and rescue cells were stained for vimentin and paxillin. Cell images were taken using a Zeiss LSM880 microscope with Airyscan. The images of vimentin filaments and paxillin staining (>1 µM in length) in cell protrusions were used for Imaris quantitative analysis. White scale bar = 10 μm, yellow scale bar = 3 μm. Imaris software was utilized to 3D-render vimentin filaments and paxillin surfaces. 3D-rendered vimentin is green, paxillin contacting vimentin is cyan, and paxillin alone is red. (B) Distance transformation was utilized to quantify the percent of vimentin filaments contacting paxillin surfaces using Imaris software. (C–E) The length of vimentin filaments, paxillin surfaces and paxillin area were quantified using Imaris software. Statistics used in all graphs were one-way ANOVA with Tukey’s post-hoc test, n = 5, *p < 0.05. (F) Representative immunoblots showing the effects of miR-509 and Plk1 on vimentin Ser-56 phosphorylation. Extracts of HASM cells treated with miR-Ctrl, miR-509 or miR-509 plus Plk1 expression construct were immunoblotted with antibodies against phospho-vimentin (Ser-56), total vimentin, and GAPDH. (G) Vimentin phosphorylation is normalized to the level obtained from cells treated with miR-Ctrl. Data are mean ± SD (n = 4, **p < 0.01). One-way ANOVA was used for statistical analysis of B–E and G.
