Fig 2: Cav-1 was negatively regulated by vimentin filaments. (A) Representative immunofluorescence images showed the subcellular distribution of endogenous vimentin and Cav-1. The white dotted outline showed the perinuclear vimentin-rich region. Bars, 10 µm. (B) The correlation analysis indicated by Pearson’s coefficient of the fluorescence signals of endogenous vimentin and Cav-1. The correlation between exogenous expressed Cav-1-mCherry and endogenous antibody stained Cav-1 was used as a positive control. (C) Representative images from time-lapse videos (Supplementary Materials) of U2OS cells expressing vimentin-GFP and Cav-1-mCherry. Magnified panels showed representative images at different time points in the yellow box of the left panel, and white arrows indicated the positions of individual Cav-1 vesicle observed in the video (Supplementary Materials). Bars, 10 µm in the left panels and 2 µm in the right magnified panels, respectively. (D) The extracts of U2OS cells with wild-type and vimentin-knockout (VIM KO) were subjected to Western blot analysis. (E) The quantified relative levels of phosphorylated Cav-1 (P-Cav-1) protein normalized to the internal control GAPDH in wild-type and vimentin-knockout cells from Western blots. The data was from five independent experiments (n = 5). (F,G) The intensity of P-Cav-1 was increased in vimentin-knockout U2OS cells and was able to recover in vimentin full-length (FL) reintroducing cells. In (F), it was showed the representative images of wild-type (arrowheads in WT and VIM KO Mix), vimentin-knockout and vimentin-knockout/full-length (arrowheads in VIM KO and FL Mix) cells. In (G), it was showed the quantification of normalized relative fluorescence intensities in wild-type (n = 21), vimentin-knockout cells (n = 13) and full-length rescue cells (n = 22). Bars, 10 µm. All the quantified data was presented as mean ± s.e.m. ** p = 0.01, *** p = 0.001 (t-test).