FIGURE 4.

siRNA-mediated silencing of Lamc1 expression inhibits stromal differentiation and cytoskeletal remodeling. MESC were transfected with 20 nm siCTRL or siRNA targeted against Lamc1 (siLAMC1) as described under “Experimental Procedures.” A, after 48 and 72 h, real time PCR was conducted using primers specific for Lamc1. Fold changes indicate gene expression levels in siLAMC1-treated MESCs relative to siCTRL-treated cells. B, Western blot analysis of Lamc1 protein in whole cell lysates of MESCs transfected with siCTRL or siLAMC1. C, relative expression of C/EBPβ, ERα, PR, PRP, and ALP mRNAs in siRNA-transfected MESC at 72 h. D, 72 h following transfection, siRNA-treated MESC were subjected to immunocytochemistry to monitor PRP protein levels. Bars, 200 μm. Western blotting of lysates of cells treated with siLAMC or siCTRL was performed using antibody against PRP. Calnexin staining indicated equal protein loading (data not shown). E, after 48 h, siRNA-treated MESC were analyzed by immunofluorescence using an antibody recognizing Lamc1 (panels a and d). DAPI-stained nuclei are shown in blue (panels b and e). Actin filaments (F-actin) were labeled with Alexa Fluor-phalloidin (panels c and f). Bars, 100 μm. All immunofluorescence experiments were conducted in two independent trials, and real time PCR measurements were conducted in three independent trials.