13 research outputs found
Morphological changes in palladin-expressing fibroblasts with and without exposure to wounding media.
<p><b>A</b>) HDF transfected with WT-palladin (WT) or FX-palladin (FX) were compared to empty vector (EV) using IF analysis of phalloidin stained cells grown in complete media or wounding media. <b>B</b>) Phalloidin-stained HDF-WT cell protrusion was magnified by electron micrography (<i>inset</i>). <b>C</b>) HDF were treated as above in (<b>A</b>) and cultured in wounding media. Phalloidin stained cells were analyzed by IF. Plots illustrate the relative surface area of cells (<i>top</i>) and average length of protrusions (<i>bottom</i>). Data are representative of two independent experiments; values are expressed as the mean ± SEM. (t-test, *, p-value<0.05; **, p-value<0.01).</p
Palladin and a stimulatory trigger enhance HDF cell migration, invasion, and degradation of extracellular matrix.
<p><b>A</b>) Migration across a transwell was compared for HDF transfected with empty vector (EV), WT-palladin (WT), FX-palladin (FX) when exposed to normal complete media or wounding media. Data shown is representative average ± SEM of three independent experiments. (t-test, *, p-value<0.05) <b>B</b>) Invasion across a matrigel-covered transwell was compared for HDF cells treated as above in (<b>A</b>) when exposed to complete media, wounding media, or conditioned media from Panc-1 cells. Data shown is representative average ± SEM of three independent experiments. (t-test, **, p-value<0.01) <b>C</b>) HDF cells were transfected as above in (<b>A</b>) and plated onto coverslips coated with Texas Red-labeled gelatin. Representative images taken via IF are shown. DAPI (blue) was used to visualize nuclei. <b>D</b>) Protein lysates from HDF cells transfected as above in (<b>A</b>) were tested for RhoA activity. Each sample was run in triplicate in two independent experiments. Error bars indicate SD. (t-test, **, p-value<0.01) <b>E</b>) Proliferation assay of HDF cells transfected as above in (<b>A</b>). Data indicates mean ± SD for three independent wells.</p
Palladin-activated fibroblasts lead Panc-1 cells through the extracellular matrix.
<p><b>A</b>) Schematic of 3-D invasion assay showing Texas Red labeled gelatin/matrigel mixture in the channel with 5 ng/ml EGF as an attractant in the left port and cells (HDF ± palladin labeled with QTracker 585 with or without Panc-1 labeled with QTracker655) seeded into the right port. Cell movement was evaluated as cells traversed the channel (right to left directional movement) using confocal microscopy. <b>B</b>) Palladin-activated fibroblasts (green) invade further into the red matrigel channel at all time points tested (24 and 72 hours) <i>(Right panels)</i> compared to HDF with EV <i>(Left panels)</i>. Representative images taken with confocal microscopy are shown. Bars indicate 100 µm. <b>C</b>) The yellow boxed region is shown at higher magnification to the right. <i>top</i>, Red matrigel is uniform in the empty channel prior to invasion. <i>middle</i>, Palladin-activated fibroblasts (green fluorescence emission) created black tunnels (devoid of Texas Red signal; see arrow) within the matrix. <i>Bottom</i>, Fibroblasts (white phalloidin stain) can move single file through the tunnels. Shown are representative images taken with confocal microscopy. Bars indicate 50 µm. <b>D</b>) Panc-1 cells (pink; see arrowheads) follow palladin-activated fibroblasts (white) through the channel while they do not follow the HDF-EV. Shown are representative images taken with confocal microscopy 72 hours after co-culture. Channels were fixed and stained with DAPI.</p
Palladin is up-regulated in normal fibroblasts co-cultured with pancreatic cancer or normal epithelial cells containing K-ras.
<p><b>A</b>) HDF cells were examined for palladin expression via IF following co-culture with pancreatic cancer cell lines, MiaPaCa or Panc-1, for 7 days. TGFβ1 is shown as a positive control for up-regulating palladin in normal fibroblasts, no epithelial cells is the negative control. Scale bars indicate 20 µm. <b>B</b>) HDF cells were examined for α-SMA or palladin expression via IF following co-culture with normal pancreatic duct epithelial cells (HPDE) that were mock transfected or transfected with wild-type or mutant K-ras. Scale bars indicate 20 µm.</p
90 kD palladin and α-SMA staining increase with progression of pancreatic tumorigenesis.
<p><b>A</b>) Expression of palladin and α-SMA was examined via IHC staining in pancreas specimens. Left: there is little to no staining in normal pancreas. Middle: expression increases in the peri-tumoral fibroblasts of pancreatic dysplasia or PanIN 2. Right: expression is highest and most widespread in the fibroblasts surrounding pancreatic cancer. <b>B</b>) Semi-quantitative readings provide IHC scores for tissue microarray staining of normal (NL), low-grade dysplasia (PanIN 2), high-grade dysplasia (PanIN 3) and cancer (CA) by palladin and α-SMA. Staining of palladin or α-SMA was scored as 0 to 4. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030219#pone.0030219.s003" target="_blank">Table S1</a>.</p
Palladin expression is sufficient to induce normal human dermal fibroblasts (HDF) cells to become myofibroblasts.
<p><b>A</b>) HDF cells transfected with 90 kD palladin (WT or FX) or empty vector (EV) were examined for the myofibroblast markers α-SMA and vimentin via IF. Red = α-SMA or vimentin; Blue = DAPI. <b>B</b>) HDF cells were treated as above in (<b>A</b>) and the expression of α-SMA was analyzed by RT-PCR. RNA was harvested at the number of days indicated post-transfection. <b>C</b>) Expression of α-SMA was analyzed by RT-PCR. GAPDH was used as an internal loading control and the relative expression of each sample versus TGF-β1 treated samples were plotted as fold induction ± SD.</p
Silencing of Palladin abrogates the myofibroblast phenotype and function.
<p><b>A</b>) HDF cells were stably transfected with control shRNA (C) or shRNA against 90 kD palladin (Pal shRNA). α-SMA expression following co-culture with Panc-1 cells was analyzed via RT-PCR. Analysis of untreated HDF (U) is shown for comparison. The first lane is a non-template negative control; GAPDH is shown as an internal loading control. Shown is data from one representative shRNA stable clone (of three independent shRNA constructs tested). <b>B</b>) Invasion across a matrigel coated transwell was compared for HDF transfected as in (<b>A</b>). Shown is the mean ±SD. Shown is data from one representative shRNA stable clone (of three shRNA constructs tested). <b>C</b>) HDF cells as in (<b>A</b>) were grown to confluence on coverslips and wounded with scratch test. Migration was assessed via IF 24 hours after wounding. At least 3 observations for each condition were analyzed. Blue = DAPI. Shown is data from one representative shRNA stable clone (of three shRNA constructs tested).</p
Palladin-activated fibroblasts use invadopodia to degrade extracellular matrix.
<p><b>A</b>) The underside of a 3 µm transwell shows podosomes/invadopodia from an HDF-WT fibroblast that has invaded through the matrigel via IF. The “feet” were severed with a razor for proteomic analysis. <b>B</b>) HDF cells transfected with GFP-empty vector (EV) or GFP-wildtype palladin (WT) were examined with phalloidin staining via IF. Palladin-expressing fibroblasts have linear protrusions (<i>arrowheads</i>) filled with proteases and lysozymes such as Cathepsin D. Shown are representative images. <b>C</b>) Proteomic analysis of the “feet” reveals overexpression of proteolytic enzymes, Rho activation proteins, and verifies the presence of proteins previously identified in invadopodia. Shown are protein ratios from fibroblasts with WT or FX palladin relative to empty vector. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030219#pone.0030219.s004" target="_blank">Table S2</a>. <b>D</b>) HDF cells transfected with wildtype palladin were grown on matrigel covered coverslips. Erosive degraded tracks were detected via electron microscopy. Bar indicates 1 µm.</p
Quantitative Glycoproteomics Analysis Reveals Changes in N‑Glycosylation Level Associated with Pancreatic Ductal Adenocarcinoma
Glycosylation plays an important
role in epithelial cancers, including
pancreatic ductal adenocarcinoma. However, little is known about the
glycoproteome of the human pancreas or its alterations associated
with pancreatic tumorigenesis. Using quantitative glycoproteomics
approach, we investigated protein N-glycosylation in pancreatic tumor
tissue in comparison with normal pancreas and chronic pancreatitis
tissue. The study lead to the discovery of a roster of glycoproteins
with aberrant N-glycosylation level associated with pancreatic cancer,
including mucin-5AC (MUC5AC), carcinoembryonic antigen-related cell
adhesion molecule 5 (CEACAM5), insulin-like growth factor binding
protein (IGFBP3), and galectin-3-binding protein (LGALS3BP). Pathway
analysis of cancer-associated aberrant glycoproteins revealed an emerging
phenomenon that increased activity of N-glycosylation was implicated
in several pancreatic cancer pathways, including TGF-β, TNF,
NF-kappa-B, and TFEB-related lysosomal changes. In addition, the study
provided evidence that specific N-glycosylation sites within certain
individual proteins can have significantly altered glycosylation occupancy
in pancreatic cancer, reflecting the complexity of the molecular mechanisms
underlying cancer-associated glycosylation events
Multiplex Targeted Proteomic Assay for Biomarker Detection in Plasma: A Pancreatic Cancer Biomarker Case Study
Biomarkers are most frequently proteins that are measured
in the
blood. Their development largely relies on antibody creation to test
the protein candidate performance in blood samples of diseased versus
nondiseased patients. The creation of such antibody assays has been
a bottleneck in biomarker progress due to the cost, extensive time,
and effort required to complete the task. Targeted proteomics is an
emerging technology that is playing an increasingly important role
to facilitate disease biomarker development. In this study, we applied
a SRM-based targeted proteomics platform to directly detect candidate
biomarker proteins in plasma to evaluate their clinical utility for
pancreatic cancer detection. The characterization of these protein
candidates used a clinically well-characterized cohort that included
plasma samples from patients with pancreatic cancer, chronic pancreatitis,
and healthy age-matched controls. Three of the five candidate proteins,
including gelsolin, lumican, and tissue inhibitor of metalloproteinase
1, demonstrated an AUC value greater than 0.75 in distinguishing pancreatic
cancer from the controls. In addition, we provide an analysis of the
reproducibility, accuracy, and robustness of the SRM-based proteomics
platform. This information addresses important technical issues that
could aid in the adoption of the targeted proteomics platform for
practical clinical utility