Nuclear uPA up-regulates expression of α-SMA in BJ cells. (A) BJ cells were serum-starved for 24 hours and serum-free medium containing 10 nM WT-scuPA (bottom panel) was then added for 24 hours. Control cells were replenished with fresh serum-free medium alone (top panel). The cells were washed, fixed in 4% PFA for 15 minutes, and permeabilized in 0.1% TritonX-100/PBS. α-SMA was visualized using mouse anti–α-SMA MAb and goat α-mouse Alexa 488-conjugated pAb (green). F-actin was detected with Alexa 647-conjugated phalloidin (red). Nuclei were stained with DAPI (blue). Images were taken using a Leica DMI 4000B microscope at 40× magnification equipped with Leica DFC350FX camera and Leica Application Suite (version 2.3.4R2) software (Leica Microsystems CMS). Scale bar represents 20 μm. (B) WT-scuPA, but not ΔK-scuPA, up-regulates α-SMA. BJ cells were serum-starved for 24 hours and serum-free medium containing either 1 to 50 nM WT-scuPA (left panel) or 1 to 50 nM ΔK-scuPA (right panel) was added for 24 hours. Cell lysates were prepared as in “Methods,” analyzed by SDS-PAGE and WB using mouse anti–α-SMA MAb and α-GAPDH MAb. The results shown are from 1 experiment representative of 5 so performed. (C) Nucleolin mediates scuPA-induced up-regulation of α-SMA. BJ cells were transduced with either control lentivirus (control) or lentivirus carrying nucleolin-targeting shRNA as described in “Methods.” Cells were starved as above and stimulated with WT-scuPA as in panel B. Expression of α-SMA and GAPDH was analyzed as in panel B. The results shown are from 1 experiment representative of 3 so performed.