TGF-β induced EMT.

<p>A549 cells were cultured in medium without (A, C) or with TGF-β (2 ng/ml) (B, D) for 3 days. Cellular morphology (A, B) was photographed by phase contrast microscope. Actin cytoskeleton (C, D) was visualized by Oregon green 488 conjugated phalloidin staining and photographed by fluorescence microscope. Scale bar = 50 µm. The effects of TGF-β on expressions of EMT markers, E-cadherin and vimentin, were examined by western blot. GAPDH from the same loading was used as control.</p

Podocalyxin is required for TGF-β induced EMT marker expressions.

<p>A549 and PODXL-KD cells were treated with TGF-β for 1–3 days, and monitored by western blot for their expressions of podocalyxin, E-cadherin and vimentin. TGF-β up-regulated the production of podocalyxin and vimentin in A549 cells, and down-regulated E-cadherin level to zero in a time dependent manner. Silencing of podocalyxin interfered with TGF-β mediated down-regulation of E-cadherin and up-regulation of vimentin.</p

Compositional analysis of Podocalyxin containing immunoprecipitates.

#<p>pep: number of peptides matched.</p><p>log(e): probability of mismatched.</p

Effects of podocalyxin silence on cell morphology and podocalyxin production.

<p><b>A</b>): Podocalyxin shRNA transduced cells (PODXL-KD) (lane 2,4,6,8) and control A549 cells (transduced with the same vector without shRNA insert) (lane 1,3,5,7) were cultured in the presence or absence of TGF-β for 1 to 3 days. Podocalyxin levels were examined by western blot. GAPDH were also determined as loading controls. <b>B</b>): Cell morphology under phase contrast microscope before and after silencing podocalyxin. Scale bar = 50 µm.</p

Induction of podocalyxin by TGF-β.

<p>A549 cells were incubated with TGF-β (2 ng/ml) for the indicated times and the cells were collected for western blot. The blots were established by mouse anti-podocalyxin and HRP-conjugated rabbit anti-mouse secondary antibody. GAPDH was also monitored on the same filter as control.</p

The distribution of podocalyxin.

<p>A549 and PODXL-KD were cultured in the presence or absence of TGF-β (2 ng/ml) for 3 days. The cells were fixed with 3.8% paraformaldehyde and permeablized. The expression of podocalyxin (red) was visualized by applying primary rabbit anti-podocalyxin antibody and cy3 conjugated goat anti-rabbit secondary antibody. F-actin was stained with Oregon green conjugated phalloidin. Images of immunofluorescence and differential interference contrast (DIC) were taken for each field and merged. The blue color in the merged pictures indicates nuclei stained by DAPI. Arrows indicated the ruffles of cell protrusions. Scale bar = 50 µm.</p

Colocalization of collagen type 1 with podocalyxin.

<p>Double immunofluorescence was performed to determine the distribution of podocalyxin (red) and collagen type 1 (green). A549 cells in chamber slide (A–D), A549 cells pretreated with TGF-β for 3 days were transferred into transwells to establishing migration (E–G), and human breast cancer cell MDA-MB-231 in chamber slide (H–J) were fixed and permeablized for following immunofluorescent staining. Rabbit anit-podocalyxin and goat anti-collagen type 1 chain 2 were visualized with Donkey anti-rabbit conjugated with cy3 and Donkey anti-goat conjugated with Oregon green 488. The merged images were also shown respectively (C, G, J). A DIC image (D) was also added to show the ruffle structure. The enrichments and colocalization of both podocalyxin and collagen were indicated by arrows.</p

Podocalyxin enriched on the leading edges of migrating cells.

<p>A549 (A–D) and PODXL-KD (E–H) cells were pretreated with TGF-β for 3 days and placed in transwell containing TGF-β (5 ng/ml) gradient for two hours. The cells on the filter were fixed with paraformaldehyde (3.8%) and stained for podocalyxin (Cy3 red) and F-actin (Oregon green-488 Phalloidin, green). Both podocalyxin and F-actin enriched on the leading edges of the migrating cells and colocalised each other as showed in merged photo (C). In contrast very few PODXL-KD cells were observed passing through the pores under the same conditions. Bright fields combined images (D and H) were also provided to visualize the locations of pores.</p

Quantification of the Host Response Proteome after Mammalian Reovirus T1L Infection

<div><p>All viruses are dependent upon host cells for replication. Infection can induce profound changes within cells, including apoptosis, morphological changes, and activation of signaling pathways. Many of these alterations have been analyzed by gene arrays to measure the cellular “transcriptome.” We used SILAC (stable isotope labeling by amino acids in cell culture), combined with high-throughput 2-D HPLC/mass spectrometry, to determine relative quantitative differences in host proteins at 6 and 24 hours after infecting HEK293 cells with reovirus serotype 1 Lang (T1L). 3,076 host proteins were detected at 6hpi, of which 132 and 68 proteins were significantly up or down regulated, respectively. 2,992 cellular proteins, of which 104 and 49 were up or down regulated, respectively, were identified at 24hpi. IPA and DAVID analyses indicated proteins involved in cell death, cell growth factors, oxygen transport, cell structure organization and inflammatory defense response to virus were up-regulated, whereas proteins involved in apoptosis, isomerase activity, and metabolism were down-regulated. These proteins and pathways may be suitable targets for intervention to either attenuate virus infection or enhance oncolytic potential.</p> </div

Kinetics of reovirus growth and viral-induced cytopathology.

<p>Each of five different cell lines (L929, A549, HEK293, CaCo₂ and Hela) were infected at MOI  = 1 PFU/cell with T1L (a) or T3D (b). Cell lysates were harvested at 0, 24, 48 and 72hpi and titrated. Experiments were performed in triplicate; error bars represent standard error. Virus titers were greatest in the L929 and HEK293 cells for both virus strains. HEK293 (c) and L929 (d) cells were then re-analyzed as in (a) and (b) after infection at MOI  = 5 and at additional time points. Aliquots of the infections in (c) and (d) were also assessed for cell viability by trypan blue exclusion (e and f, respectively), with 100 μg/ml puromycin used as a positive cell killing control. Experiments were performed in duplicate; error bars represent standard error.</p