Vimentin phosphorylation is required for β1 integrin expression, activation, and cell spreading. A: Alexa-488 phalloidin staining of control (far left), vimentin-knockdown (left), bisindolylmaleimide (BIM)-treated (center), calphostin C-treated (right), and PKC-ε-knockdown cells (far right) spreading on collagen. Bar = 30 μm. B: Western blot illustrates knockdown of PKC-ε by siRNA. C: histogram showing differences in mean number of cell extensions ± SE between control, vimentin-knockdown, BIM-treated, calphostin C-treated, and PKC-ε-knockdown cells spreading on collagen. D: flow cytometry indicates decreased cell surface expression of β1 integrins following BIM treatment (left) or knockdown of PKC-ε (right). E: histogram shows mean 4B4 fluorescence intensity ± SD in control, vimentin-knockdown, BIM-treated, calphostin C-treated, and PKC-ε-knockdown cells. F: flow cytometry indicates decreased β1 integrin activation following BIM treatment (left) or knockdown of PKC-ε (right). G: histogram shows mean % of 12G10 fluorescence intensity ± SD among control, vimentin-KD, BIM-treated, calphostin C-treated, and PKC-ε-KD cells.