The monolayer was scratched with a pipette tip and washed with PBS to remove floating cells

The monolayer was scratched with a pipette tip and washed with PBS to remove floating cells. imaging was carried out using an inverted microscope (IX 71, Olympus, Japan) with a total internal reflective fluorescence illuminator, a 100X/1.45NA Plan Apochromat TIR objective (Olympus, Tariquidar (XR9576) Japan) and a 14-bit back-illuminated electron-multiplying charge-coupled device (EMCCD) (Andor iXon DU-897 BV) [12, 21, 22]. A 488-nm argon laser with the power of 5 mW (Melles Griot, Carlsbad, CA) was used to excite the GFP in epi-fluorescence mode. Before being directed into the EMCCD (gain: 300) the fluorescent signals were passed through two filters, BA510IF and HQ 525/50 (Chroma Technology). To insure homogeneous illumination only the central quarter of the chip (256256 pixels) was used for imaging analysis. MetaMorph software (Molecular Device) was used to acquire and analyze movies (200 frames for each sample at a frame rate of 10 Hz). The background fluorescence was first subtracted from each frame using the rolling ball method in Image J software (National Institute of Health). The first frame of each movie was used for the selection of fluorescent spots and the threshold was set a four times that of the mean intensity of an area lacking fluorescent spots. The image was then filtered again with a user-defined program in Matlab. 4.5. Western Blotting Western blotting was performed in order to study downstream signaling of the TGF- pathway. For detection of Smad3, the starved HeLa cells were treated with vehicle (0.1% DMSO) or hesperetin (100M) for 1 hour in the absence or presence of TGF-1. For the detection of p21, the starved HeLa cells were treated with vehicle (0.1% DMSO) or hesperetin (100M) for 24 hours in the absence or presence of TGF-1. Cells were resuspended in 200 l of Tariquidar (XR9576) 0.9% saline with protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 10 g/ml aprotinin, 10 g/ml leupeptin, and 10 g/ml pepstatin A) and mixed with 200 l double-strength Laemmli buffer (100 mM Tris-HCl, pH 6.8, 200 mM dithiothreitol, 4% SDS, 0.2% bromophenol blue, and 20% glycerol). Cell lysates were denatured for 10 minutes at 95C. Cell lysates were were separated by SDS Tariquidar (XR9576) gel electrophoresis (7.5% SDS gels) and transferred Tariquidar (XR9576) to COPB2 polyvinylidene fluoride (PVDF membranes) with 0.192 M glycine, 0.025 M Tris and 20% methanol. Membranes were saturated for one hour at 4C in TBS, pH 7.4, containing 5% nonfat milk and probed for two hours at room temperature with primary antibodies for p-Smad3 (Cell Signaling, dilution 1:1000), Smad3 (Cell Signaling, dilution 1:1000) or p21 (Cell Signaling, dilution 1:1000). After washing the blots were probed for one hour at room temperature with a peroxidase-conjugated secondary antibodies. Antibody binding was detected by enhanced chemiluminescence (Amersham Life Sciences, Amersham, UK). 4.6. Immunofluorescence Imaging HeLa cells were cultured in 35 mm dishes for a period of 24 hours, followed by washing with PBS, and incubation with fresh serum-free medium for another 24 hours. The nuclear translocation of Smad3 was assessed by treating cells with 0.1% DMSO (vehicle) or hesperetin (100 M) for 1 hour, in the absence or presence of TGF-1. Cells were then fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. 1% BSA in PBST (PBS containing 0.1% Tween-20) was used to block nonspecific binding sites. The fixed cells were then incubated with Smad3 monoclonal rabbit primary antibody overnight (Cell Signaling, 1:200 dilution), followed by washing and a 1 hour incubation with Alexa 488-conjugated anti-rabbit IgG secondary antibody (Invitrogen, 1:500 dilution). All antibodies were diluted in PBST solution containing 1% BSA. Fluorescence imaging.

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