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

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

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

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

Peptide Retention Time Prediction in Hydrophilic Interaction Liquid Chromatography: Data Collection Methods and Features of Additive and Sequence-Specific Models

The development of a peptide retention prediction model for hydrophilic interaction liquid chromatography (XBridge Amide column) is described for a collection of ∼40 000 tryptic peptides. Off-line 2D LC-MS/MS analysis (HILIC-RPLC) of <i>S. cerevisiae</i> whole cell lysate has been used to acquire retention information for a HILIC separation. The large size of the optimization data set (more than 2 orders of magnitude compared to previous reports) permits the accurate assignment of hydrophilic retention coefficients of individual amino acids, establishing both the effects of amino acid position relative to peptide termini and the influence of peptide secondary structure in HILIC. The accuracy of a simple additive model with peptide length correction (<i>R</i>² value of ∼0.96) was found to be much higher compared to an algorithm of similar complexity applied to RPLC (∼0.91). This indicates significantly smaller influence of peptide secondary structure and interactions with counterions in HILIC. Nevertheless, sequence-specific features were found. Helical peptides that tend to retain stronger than predicted in RPLC exhibit negative prediction errors using an additive HILIC model. N-cap helix stabilizing motifs, which increase retention of amphipathic helical peptides in RP, reduce peptide retention in HILIC independently of peptide amphipathicity. Peptides carrying multiple Pro and Gly (residues with lowest helical propensity) retain stronger than predicted. We conclude that involvement of the peptide backbone’s carbonyl and amide groups in hydrogen-bond stabilization of helical structures is a major factor, which determines sequence-dependent behavior of peptides in HILIC. The incorporation of observed sequence dependent features into our Sequence-Specific Retention Calculator HILIC model resulted in 0.98 <i>R</i>² value correlation, significantly exceeding the predictive performance of all RP and HILIC models developed for complex mixtures of tryptic peptides

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

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

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

Response of Primary Human Airway Epithelial Cells to Influenza Infection: A Quantitative Proteomic Study

Influenza A virus exerts a large health burden during both yearly epidemics and global pandemics. However, designing effective vaccine and treatment options has proven difficult since the virus evolves rapidly. Therefore, it may be beneficial to identify <i>host</i> proteins associated with viral infection and replication to establish potential new antiviral targets. We have previously measured host protein responses in continuously cultured A549 cells infected with mouse-adapted virus strain A/PR/8/34­(H1N1; PR8). We here identify and measure host proteins differentially regulated in more relevant primary human bronchial airway epithelial (HBAE) cells. A total of 3740 cytosolic HBAE proteins were identified by 2D LC–MS/MS, of which 52 were up-regulated ≥2-fold and 41 were down-regulated ≥2-fold after PR8 infection. Up-regulated HBAE proteins clustered primarily into interferon signaling, other host defense processes, and molecular transport, whereas down-regulated proteins were associated with cell death signaling pathways, cell adhesion and motility, and lipid metabolism. Comparison to influenza-infected A549 cells indicated some common influenza-induced host cell alterations, including defense response, molecular transport proteins, and cell adhesion. However, HBAE-specific alterations consisted of interferon and cell death signaling. These data point to important differences between influenza replication in continuous and primary cell lines and/or alveolar and bronchial epithelial cells